Microparticles for cell disruption and/or biomolecule recovery

ABSTRACT

The present invention provides novel methods of cell disruption and release of biomolecules from a cell. The invention comprises the use of positively and/or negatively charged microparticles comprising ground resin. It is particularly useful for purification of biomolecules from cell culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Non-Provisionalapplication Ser. No. 14/913,337, filed Feb. 19, 2016, which is aNational Phase Patent Application of International Patent ApplicationNumber PCT/EP2014/068015, filed on Aug. 25, 2014, which claims priorityto European Patent Application No. 13181540.9, filed on Aug. 23, 2013and European Patent Application No. 13181537.5, filed on Aug. 23, 2013,all of which are hereby incorporated by reference.

FIELD OF INVENTION

The present invention generally relates to the field of biomoleculerecovery and cell disruption, and particularly to the recovery ofintracellular biomolecules such as polypeptides or polynucleotides fromcell suspensions. It covers methods of recovering biomolecules fromfluids such as biological fluids. In a further aspect, the presentinvention is related to the field of cell culture and purification ofbiomolecules from cell culture.

BACKGROUND OF THE INVENTION

Despite all previous efforts to develop artificial production systems,biological entities such as cells are unrivalled in their ability toproduce complex substances such as antibiotics, proteins and nucleicacids. Nearly all substances of cellular origin produced industriallytoday are extracellular products that are produced within the cell andsubsequently excreted into the environment. However, a large proportionof potentially useful substances remain intracellular. In order torelease the intracellular material, cells are typically disintegrated bymechanical, physical, chemical or enzymatic means. The recoveryefficiency of valuable cellular products is closely linked to theirformation, location and their interactions within the biological system.In the past decades, investigations of molecular structures and theirfunctionality within these cellular systems have enhanced thediversification of such products and their large-scale processing. Atthe same time, requirements regarding product quality, especially in thehealthcare sector, resulted in the necessity to deliver well defined,effective and highly pure substances.

Current methods for cell disruption include mechanical andnon-mechanical methods. Non-mechanical methods comprise physical(decompression, osmotic shock, thermolysis, freeze drying, microwave),chemical (antibiotics, chelating agents, detergents, solvents, alkalis,supercritical CO₂) and enzymatic (lysis, autolysis, phages) methods. Onthe other hand, mechanical approaches and devices for cell disruptioncomprise bead milling, homogenisers, cavitation (ultrasonic,hydrodynamic) and microfluidizers. Only some cell disruption methods(mainly mechanical) are performed at an industrial scale, where they arecommonly integrated in downstream (e.g. recovery, purification)processing.

For large scale, the most common mechanical methods are bead milling andhigh pressure homogenisation which are typically implemented as standardoperations. When using high pressure homogenisation, a cell solution isforced through a narrow valve under high pressure. By passing throughthe valve, cells are subjected to turbulence, cavitation, high shearforces and a sudden pressure drop upon discharge which tear the cellsapart. The stress and erosion of the valve must be considered in theconstruction and increase with the rising homogenising pressure.Further, the temperature of the solution rises with increasing pressure,rendering the method energy-consuming and making cooling of the deviceand the suspension necessary, particularly when temperature-labileenzymes are released. Approximately more than 90% of the power consumedby the homogeniser dissipates as heat, and the cooling cost represents alarge portion of the total costs for cell disintegration. Further,successive passages are often required in order to achieve sufficientlyhigh yields (Kula et al., “Purification of Proteins and the Disruptionof Microbial Cells.” Biotechnology Progress 1987).

In bead milling, beads are added to a biological fluid which issubsequently subjected to high speed agitation by stirring or shaking.By collision with beads cells are disrupted and intracellular contentsare released. Bead milling also produces heat and requires cooling. Itis a rather complex process influenced by a variety of parameters,including construction of the bead mill, operational parameters andproduct specific properties. Construction and geometry of the bead millare crucial process variables. However, smaller versions are oftengeometrically dissimilar to industrial-scale bead mills, whichcomplicates the extrapolation of data from laboratory trials to batchperformance (Kula et al., “Purification of Proteins and the Disruptionof Microbial Cells.” Biotechnology Progress 1987).

Mechanical disruption methods suffer from several drawbacks. Becausecells are entirely disrupted, all intracellular materials are released,thereby increasing the contaminants content of the intermediate product.Thus, the product of interest must be separated from a complex mixtureof proteins, nucleic acids, and cell fragments. In addition, releasednucleic acids may increase the viscosity of the solution and maycomplicate subsequent processing steps such as chromatography. The celldebris produced by mechanical disintegration often consists of smallcell fragments, making the solution difficult to clarify. Completeproduct release often requires more than one pass through the disruptiondevice, which exacerbates the problem by further reducing the size ofthe fragments. These are difficult to be removed by continuouscentrifugation, because the throughput of the device is inverselyrelated to the square of the particle diameter. Filtration iscomplicated by the sticky nature of the homogenate and by its tendencyto foul membranes. Furthermore, mechanical methods require regularmaintenance and costly equipment and are energy consuming. They generateheat and require extensive cooling in order to be usable fortemperature-sensitive enzymes. Further, they expose the cells andtherefore the extracted products to high shear stress. Most productswill be denatured by the heat generated unless the device issufficiently cooled.

As mentioned above, more selective release methods involve physical,chemical or enzymatic treatment. Chemical treatment involves the use ofEDTA, chaotropic agents, organic solvents, antibiotics, acids, alkalisand surfactants. Besides the problem of waste disposal of excesschemicals, these methods are rather expensive and thus not suitable forlarge-scale application. Contamination of the desired product with thechemicals is another drawback. Some chemicals are not very selective andtend to damage sensitive proteins, enzymes and the cells walls.

Enzymatic cell disruption is more specific but is often limited whenapplied to complex cell structures with several distinct layers such asbacterial cell walls. Further, it is restricted by the cost of enzymeand buffers. Thus, enzymatic treatment is not applicable at large orindustrial scale either. Another drawback is the potential contaminationof the desired product with the enzyme.

Physical permeabilization can be accomplished by freeze-thawing orosmotic shock treatment. With freeze-thawing, multiple cycles arenecessary for efficient product release, and the process can be quitelengthy. Osmotic shock treatment, on the other hand, may not besufficient to disrupt cells with robust cell wall structures.

In sum, disadvantages of these methods include comparably high costs,low practicability at large scale, low efficiency and reproducibility,and the necessity to remove added substances after the release.

Taking into account the potential of cellular production systems, thereis a need for alternative methods for recovering biomolecules, inparticular from cell suspensions, which are easy to handle,cost-efficient, and scalable. Further, the methods should be gentleenough for sensitive biomolecule products and enable a highly selectivebiomolecule recovery which yields a product with low contamination. Itis therefore one objective of the present invention to provide a methodor system which overcomes one or more of the above mentioned drawbacks.

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange. It includes, however, also the concrete number, e.g., about 20includes 20.

The term “less than” or “greater than” includes the concrete number. Forexample, less than 20 means less than or equal to. Similarly, more thanor greater than means more than or equal to, or greater than or equalto, respectively.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”.

When used herein, “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms.

It should be understood that this invention is not limited to theparticular methodology, protocols, material, reagents, and substances,etc., described herein and as such can vary. The terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims.

All publications and patents cited throughout the text of thisspecification (including all patents, patent applications, scientificpublications, manufacturer's specifications, instructions, etc.),whether supra or infra, are hereby incorporated by reference in theirentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material.

SUMMARY

The present invention provides novel methods for obtaining biomolecules,also referred to as biomolecule recovery which overcomes one or more ofthe above mentioned drawbacks. The method can be used to obtainbiomolecules from liquids and fluids, such as cell cultures, cellhomogenates, cell lysates, cell suspensions, fermentation broth, culturebroth, fermentation supernatant, culture supernatant, cell supernatants,such as from E. coli, Pichia pastoris, and CHO cell culture. Further,the present invention involves methods of cell disruption which can beused to release biomolecules contained in cells. As will be shown in thedescription, the present invention is particularly useful for thedisruption of cells and recovery of intracellular biomolecules. Themethods disclosed herein are simple, cost-efficient, and readilyscalable for industrial application. The methods provide a simpleprocess for selective recovery of biomolecules, where neither complexequipment nor soluble additives, excepting buffer and salt, arerequired. As described earlier, prior art methods for biomoleculerecovery from cell suspensions involve either mechanical stress and/orchemical additives. In contrast to conventional methods applied atindustrial scale today, the methods of the present invention are gentleand do not subject the cells and/or the desired biomolecule products toharsh conditions. Thus, the methods disclosed herein reduce the amountof contaminating debris and exert less potentially harming mechanicaland physical stress on the desired biomolecules.

The inventors have surprisingly discovered that charged microparticlesor hydrophobic microparticles comprising ground resin can act to recover(herein also referred to as “to extract”) biomolecules from biologicalfluids. Further, in some embodiments, when added to a cell suspension,charged microparticles can disrupt cells and at the same time adsorb thebiomolecules released from the cells. Therefore, in one aspect themicroparticles can be used to disrupt cells and release biomolecules andfurther adsorb the biomolecules in a simple and scalable manner. In someaspects, the microparticles have been found to form flocs uponinteraction with cells and/or biomolecules and/or counter chargedmicroparticles and can be easily separated from the biological fluid.The released biomolecules can thus easily be recovered by separating theflocs with biomolecules adsorbed thereon. The present invention thusfurther provides simplified methods where the steps of cell disruptionand biomolecule extraction in the downstream processing are combined.This technique is termed colloidal solid phase extraction (CSPE).Without being bound by theory, it is assumed that positively chargedmicroparticles derived from an anion-exchange resin build flocs withcells by adsorbing to the negatively charged cell surface, therebydisplacing cations, in particular calcium and/or magnesium ions, whichare associated with the usually negatively charged cell surface ofparticularly bacterial cells. As a result, the lining up of themolecules building the cell membrane becomes somehow destabilized andthus cell become leaky.

In case of a negatively charged particles derived from a chelatingcation exchange resin, it is assumed that such particles bind cations,in particular calcium and/or magnesium, thereby destabilizing the liningup of the molecules building the cell membrane. For hydrophobicmicroparticles, it was also found that they form flocs.

This is currently a unique method for extraction of intracellularbiomolecules by gently “opening” the cell with low fragmentation of thecellular structures (e.g. cell walls), resulting in a reduction incontaminants levels. At the same time, this novel technique allows forselective biomolecule extraction, and is comparable to mechanicaldisruption methods known in the art, e.g. high pressure homogenisationand bead milling, in terms of efficiency, but offers a higherselectivity and a lower contamination level. At the same time, using thenovel method disclosed herein, it is possible to maintain the integrityof the cells while the desired biomolecule is recovered. Thus, “opening”cells means that cells, however are preferably not entirely disrupted.Accordingly, the “opening” preferably means that cells become leaky andthus their cytoplasmic contents leak out.

In a first aspect, the invention provides the novel use of positivelycharged microparticles and/or negatively charged microparticles forbiomolecule recovery, wherein the positively charged microparticlescomprise ground polymeric anion-exchange resin, and wherein thenegatively charged microparticles comprise ground polymeric cationexchange resin. In one embodiment, only positively chargedmicroparticles are used. In another embodiment, only negatively chargedmicroparticles are used. In a further embodiment, both positivelycharged microparticles and negatively charged microparticles are used.

In a second aspect of the invention, the composition compriseshydrophobic microparticles. These hydrophobic microparticles are capableof adsorbing in particular peptides or polypeptides, but also otherbiomolecules. The mechanism for adsorption is thought to be basedprimarily on hydrophobic (Van der Weals, London Type) attractionsbetween the hydrophobic portions of the adsorbed ligands such aspeptides or polypeptides and the polymeric surface of themicroparticles.

The microparticles are obtainable (can be obtained) by grinding resin asdescribed herein, for example, by grinding ion-exchange resin andoptionally conditioning the ground resin. Such particles are referred toherein as “microparticles,” “adsorbent particles”, “adsorbent”,“particles”, “ground particles”, or “ground resin”. These terms are usedinterchangeably. Preferably, the microparticles are obtained by grindingconventional large-diameter small-pore particles which are usuallyintended for water de-ionization and waste water treatment.

The positively charged microparticles can be prepared by grinding anionexchange resin. The anion-exchange resin can be weakly or stronglybasic. Likewise, cation-exchange resin can be used to prepare thenegatively charged microparticles. The cation exchange resin can beweakly or strongly acidic. In some embodiments of the present invention,the cation-exchange resin and/or the anion-exchange resin can be achelating resin

The ion-exchange resin according to the present invention can be basedon any suitable material. Preferably, the resin is polystyrene-based,Hydroxyethyl methacrylate (HEMA)-based, dimethylamino ethylmethacrylate(DMAEMA)-based, dimethylamino ethylmethacrylate (pDMAEMA) based,polyacrylamide based or methacrylic acid (MAA) based. More preferably,the resin is polystyrene cross-linked with divinylbenzene (DVB).

Preferably, the microparticles are in the form of ground particleshaving an average particle size less than about 10 μm, such as less thanabout 5 μm.

Microparticles according to the present invention can be obtained bygrinding anion-exchange resin or cation exchange resin. Anion-exchangeresin may be, for example, AMBERLITE® IRA-400, AMBERLITE® IRA-485,DOWEX® 1×2-100, DOWEX® 1-8-100, DIAION® SA 20A, MARATHON® A2 or otheranion-exchange resin known in the art. Cation exchange resin may be, forexample, AMBERLITE® IRC-748, DOWEX® 50 WX2-100, DOWEX® 50 WX8-100,DIAION® SK 110, MARATHON® MSC, or other cation-exchange resin known inthe art. Preferred anionic exchange resins include AMBERLITE® IRA-458and MARATHON® A2. Preferred cationic exchange resins include AMBERLITE®IRC-748.

In another aspect, the present invention provides the use of positivelycharged microparticles and/or negatively charged microparticles orhydrophobic microparticles disclosed herein to adsorb biomolecules,preferably proteins or polynucleotides, such as DNA, e.g., plasmid DNA,cosmid DNA, BAC DNA, YAC DNA, mini-circle DNA from a fluid. The fluid isa biological fluid such as cell homogenate, fermentation supernatant,fermentation broth, culture broth, culture supernatant, cell lysate or acell suspension.

Further, the present invention provides the use of the microparticlesdisclosed herein for cell disruption. Preferably, the disrupted cellsrelease biomolecules which adsorb to the microparticles. The cells fordisruption include eukaryotic and prokaryotic cells. In one embodiment,the cell is a prokaryotic cell. The cell can be selected from the groupincluding, but not limited to, Enterobacteriaceae, Pseudomonaceae,Lactobacteriacea, or Bacillaceae. In one preferred embodiment, the cellis E. coli.

In a further aspect, the present invention provides a method forobtaining biomolecule, comprising adding the positively chargedmicroparticles and/or negatively charged microparticles or hydrophobicmicroparticles described herein to a biological fluid and recovering thebiomolecules from the biological fluid. In some embodiments, the methodfurther comprises allowing the microparticles to form flocs, removingthe flocs from the biological fluid, and desorbing the biomolecules fromthe flocs. In some instances, depending on the biomolecule to berecovered, the microparticles can be used to form flocs with unwantedcellular structure and the biomolecule is recovered in the fluid afterthe flocs are removed. The flocs can be removed from the biologicalfluid by, for example, centrifugation or filtration. In one embodiment,the biological fluid is agitated during and/or after adding themicroparticles and/or during desorbing the biomolecules from the flocsor the biological fluid.

The present invention further provides a method of disrupting cells,comprising adding charged and/or hydrophobic microparticles to a cellsuspension. Said charged microparticles can be positively charged and/ornegatively charged. In some aspects, the method further comprisesreleasing biomolecules from the cells. In addition, the presentinvention also provides a biological fluid and positively chargedmicroparticles and/or negatively charged microparticles or hydrophobicmicroparticles.

The exact nature of this invention, as well as its advantages, willbecome apparent to a skilled person from the following description andexamples. The present invention is not limited to the disclosedpreferred embodiments or examples. A skilled person can readily adaptthe teaching of the present invention to create other embodiments andapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic chemical structure of functional groups and theirbinding sites to polymer matrix for MARATHON® MSC (MMSC), MARATHON® A2(MA2), AMBERLITE® IRC748 and AMBERLITE® IRA458.

FIG. 2: Influence of the volumetric cell concentration and volumetricresin:cells ratio of MARATHON® A2 microparticles on GFP recovery fromGFP expressing E. coli at 1 hour incubation (50 mM TRIS, pH 8.0).

FIG. 3: Influence of the volumetric resin:cells ratio of MARATHON® A2microparticles on GFP recovery from GFP expressing E. coli (10% v/v) at1 hour incubation (50 mM TRIS, pH 8.0).

FIG. 4A is a graph showing the extraction kinetic of MARATHON® A2microparticles on GFP recovery from cell broth containing GFP expressingE. coli after incubation at pH 7.5. FIG. 4B is a graph showing theextraction kinetic of MARATHON® A2 microparticles on GFP recovery fromcell broth containing GFP expressing E. coli after incubation at pH 8.0.FIG. 4C is a graph showing the extraction kinetic of MARATHON® A2microparticles on GFP recovery from cell broth containing GFP expressingE. coli after incubation at pH 8.5. All experiments were performed usinga 0.2-3 hour static incubation with the microparticles.

FIG. 5: Influence of salt concentration on recovery of GFP from GFPexpressing E. coli (10% v/v) after 1 h incubation (50 mM TRIS, pH 8.0)with MARATHON® A2 microparticles (100%, 70%, 50%, 30%, 0% volumetricration of resin:cells) and different NaCl concentrations (500 mM, 100mM, 50 mM, 0 mM) and elution in 1 M NaCl.

FIG. 6: Equilibrium capacities of GFP extracted from E. coli cells (10%v/v) by adsorption on MARATHON® A2 microparticles (100%, 70%, 50%, 30%,0% volumetric ratio resin:cells) after static incubation (50 mM TRIS, pH8.0) in different NaCl concentrations (500 mM, 100 mM, 50 mM, 0 mM) andelution in 1 M NaCl. GFP from aqueous phase was quantified byfluorescence before and after elution in 1M NaCl. Difference of GFPquantity was considered to be adsorbed on resin. Trends were fitted withthe standard “Langmuir” equation.

FIG. 7A: Influence of incubation conditions on recovery of GFP from GFPexpressing E. coli (5% v/v) after stirring (1-2 hours, 50 mM TRIS, pH8.0) with chelating AMBERLITE® IRC 748 microparticles (70%, 30%volumetric ratio of resin:cells) with elution in 1 M NaCl. FIG. 7B:Influence of incubation conditions on recovery of GFP from GFPexpressing E. coli (5% v/v) after stirring (1-2 hours, 50 mM TRIS, pH8.0) with chelating AMBERLITE® IRC 748 microparticles (70%, 30%volumetric ratio of resin:cells) without elution in 1 M NaCl.

FIG. 8: Influence of volumetric resin:cells ratio and elution on GFPrecovery from GFP expressing E. coli (5% v/v) after 2 hour staticincubation (50 mM TRIS, pH 8.0) with chelating AMBERLITE® IRC748microparticles (100%, 70%, 30% volumetric resin:cells ration) and withor without elution in 1 M NaCl.

FIG. 9 Influence of volumetric cell concentration (20%, 15%, 10%, 5%v/v) of GFP expressing E. coli on GFP recovery after 2 hour stirringincubation (50 mM TRIS, pH 8.0) with chelating AMBERLITE® microparticles(70% volumetric resin:cells ratio).

FIG. 10: GFP extraction kinetic of GFP expressing E. coli at differentvolumetric cell concentrations (20%, 15%, 10%, 5% v/v) after 2 hourstirring incubation (50 mM TRIS, pH 8.0) with chelating AMBERLITE®microparticles (70% volumetric resin:cells ratio).

FIG. 11A shows the SOD recovery in the absence of acrylic IRA 458. FIG.11B shows the SOD recovery using 50% v/v resin:cells ratio of acrylicIRA 458. FIG. 11C shows the SOD recovery using 75% v/v resin:cells ratioof acrylic IRA 458. FIG. 11D shows the SOD recovery using 100% v/vresin:cells ratio of acrylic IRA 458. SOD amount was determined bySDS-Page densitometry after 1-3 hours static incubation in 50 mM TRIS(pH 8.0) at various concentrations of NaCl (1.0 M, 0.5 M, and 0 M).

FIG. 12: Recovery of SOD from E. coli homogenate obtained from 10% v/vcell suspension after short mixing and incubation (50 mM TRIS, pH 7.7)with acrylic AMBERLITE® IRA 458 microparticles (100%, 70%, 50% v/vresin:cells ratio) and elution with different NaCl concentrations (1.0M, 0.5 M, 0 M). SOD amount was determined by SDS-Page densitometry.

FIG. 13: Extraction of non-target proteins by 3 hour incubation (50 mMTRIS, pH 8.0) of GFP expressing E. coli with MARATHON® A2 microparticles(50% volumetric resin:cells ratio) after elution with different NaClconcentrations (200-1000 mM) in comparison to E. coli homogenate andhomogenate supernatant determined by SDS-Page and Coomassie staining.

FIG. 14: Densitometry analysis of the SDS-Page from FIG. 13.

FIG. 15: Protein profile of GFP expressing E. coli homogenatesupernatant in comparison to eluate obtained from E. coli by 3 hourstatic incubation (50 mM TRIS, pH 8.0) with MARATHON® A2 microparticles(50% volumetric resin:cells ratio) and elution with 300 mM NaCl.

FIG. 16A shows the dsDNA reduction kinetics during extraction of protein(SOD) using 50% v/v resin:cells of AMBERLITE® IRA458. FIG. 16B shows thedsDNA reduction kinetics during extraction of protein (SOD) using 70%v/v resin:cells of AMBERLITE® IRA458. FIG. 16C shows the dsDNA reductionkinetics during extraction of protein (SOD) using 1000% v/v resin:cellsof AMBERLITE® IRA458. FIG. 16D shows the dsDNA reduction kinetics duringextraction of protein (SOD) using 50% v/v resin:cells of MARATHON® A2.FIG. 16E shows the dsDNA reduction kinetics during extraction of protein(SOD) using 70% v/v resin:cells of MARATHON® A2. FIG. 16F shows thedsDNA reduction kinetics during extraction of protein (SOD) using 100%v/v resin:cells of MARATHON® A2. All experiments used E. coli homogenateand cell suspension (10% v/v) after static incubation (50 mM TRIS, pH8.0) with the various microparticle resins and subsequent elution withNaCl. DNA amount was estimated with “Pico Green QuantIt” assay fromInvitrogen.

FIG. 17: Endotoxin reduction during extraction kinetic of protein (SOD)from E. coli homogenate and cell suspension (10% v/v) with MARATHON® A2microparticles after static incubation (50 mM TRIS, pH8.0) andsubsequent elution with NaCl. Endotoxin amount was estimated byPyroGene™ Recombinant Factor C Assay purchased from Lonza.

FIG. 18: Atomic force microscopy (AFM) of ground resins. Heightmeasurement of MARATHON® A2 (Image 1), MARATHON® MSC (Image 2) andAMBERLITE® IRC748 (Image 3).

FIG. 19: Atomic force microscopy (AFM) of E. coli cells. Heightmeasurement of cells before (left side) and after 1 h incubation withground MARATHON® A2.

FIG. 20: Overlaid images of E. coli HMS174 (GFPmut3.1) withoutmicroparticles (picture 1), after 1 h incubation with ground MARATHON®A2 (picture 2) and with ground AMBERLITE® IRC748 (picture 3) at pH8.0 in50 mM TRIS.

FIG. 21: Viability of E. coli cells (10% v/v) after 2 hours of staticincubation (50 mM TRIS, pH8.0) with MARATHON® A2 microparticles (70%volumetric resin:cells ratio) and 1:10 dilution in physiological buffer.Live (green)/dead (red) cell staining was performed with “BacLite”fluorometric staining kit from Invitrogen.

FIG. 22: SDS-Gel shows an elution profile using microparticles tocapture GFP from cell homogenate.

FIG. 23: SDS-Gel shows an elution profile using microparticles to forrecovering IFN-γ from cell homogenate.

FIG. 24: Bar graph showing the GFP amount in the capture supernatant (1)and wash buffer (2), eluted GFP (3) in Example 13.2 compared withmaximum GFP using chemical disruption method (4).

ITEMS OF THE INVENTION

The present invention can also be characterized by the following items:

1. Use of positively charged microparticles and/or negatively chargedmicroparticles for biomolecule recovery, wherein the positively chargedmicroparticles comprise ground polymeric anion-exchange resin, andwherein the negatively charged microparticles comprise ground polymericcation exchange resin, wherein the biomolecule is preferably apolypeptide or a polynucleotide.

2. Use of a positively charged microparticles and/or negatively chargedmicroparticles for cell disruption, wherein the positively chargedmicroparticles comprise ground polymeric anion-exchange resin, andwherein the negatively charged microparticles comprise ground polymericcation exchange resin.

3. Use of hydrophobic microparticles for biomolecule recovery.

4. The use of item 1 or 2, wherein the cation exchange resin is weaklyor strongly acidic.

5. The use of item 1 or 2, wherein the anion-exchange resin is weakly orstrongly basic.

6. The use of item 1 or 2, wherein the cation exchange resin and/or theanion-exchange resin is a chelating resin.

7. The use of any one of the preceding items, wherein the anion-exchangeresin and the cation exchange resin is polystyrene-based, Hydroxyethylmethacrylate (HEMA)-based, dimethylamino ethyl methacrylate(DMAEMA)-based, dimethylamino ethyl methacrylate (pDMAEMA),polyacrylamide based, methacrylic acid (MAA)-based.

8. The use of any one of the preceding items, wherein the cationexchange resin and anion-exchange resin is polystyrene cross-linked withdivinylbenzene.

9. The use of any one of the preceding items, wherein the microparticleshave an average particle size of less than about 5 μm.

10. The use of any one of the preceding items, wherein the positivelycharged microparticles or negatively charged microparticles areobtainable by grinding a polymeric anion-exchange and/or cation-exchangeresin.

11. The use of any one of the preceding items, wherein theanion-exchange resin is AMBERLITE® IRA-400, AMBERLITE® IRA-485, DOWEX®1×2-100, DOWEX® 1-8-100, MARATHON® A2 or DIAION® SA 20A.

12. The use of any one of the preceding items, wherein the cationexchange resin is AMBERLITE® IRC-748, DOWEX® 50 WX2-100, DOWEX® 50WX8-100, MARATHON® MSC or DIAION® SK 110.

13. The use of any one of the preceding items, wherein the resin isnon-porous.

14. The use of any one of the preceding items, wherein the cell is aeukaryotic or prokaryotic cell.

15. The use of any one of items 1 to 14, wherein the cell is selectedfrom Enterobacteriaceae, Pseudomonaceae, Lactobacteriacea, orBacillaceae.

16. The use of item 14 or 15, wherein the cell is E. coli.

17. The use of any one of the preceding items, wherein the biomoleculeis a protein or a polynucleotide.

18. A method of obtaining biomolecules from a biological fluidcomprising a) adding a positively charged microparticles and/ornegatively charged microparticles or hydrophobic microparticles asdefined in any one of items 1 to 13 to a biological fluid, andrecovering the biomolecules from the biological fluid.

19. The method of item 18, further comprising: b) allowing themicroparticles to form flocs c) removing the flocs from the biologicalfluid and d) desorbing the biomolecules.

20. The method of item 19, wherein the biological fluid is a cellsuspension, fermentation broth, culture broth, a cell homogenate orfermentation supernatant.

21. The method according to any of items 18 to 20, wherein the methodfurther comprises agitating the biological fluid after step a) and/ord).

22. The method according to any of items 18-21 wherein step c) iscarried out by a separation technique, such as centrifugation orfiltration.

23. The method according to any of items 18-22, wherein the cationexchange resin is weakly or strongly acidic.

24. The method according to any of items 18-22, wherein theanion-exchange resin is weakly or strongly basic.

25. The method according to any of items 18-22, wherein the cationexchange resin and/or the anion-exchange resin is a chelating resin.

26. The method according to any of items 18-25, wherein theanion-exchange resin and the cation exchange resin is polystyrene-based,Hydroxyethyl methacrylate (HEMA)-based, dimethylamino ethyl methacrylate(DMAEMA)-based, dimethylamino ethyl methacrylate (pDMAEMA),polyacrylamide based, methacrylic acid (MAA)-based.

27. The method according to any of items 18-26, wherein the cationexchange resin and anion-exchange resin is polystyrene cross-linked withdivinylbenzene.

28. The method according to any of items 18-28, wherein themicroparticles have an average particle size of less than about 5 μm.

29. The method according to any of items 18-28, wherein the positivelycharged microparticles or negatively charged microparticles areobtainable by grinding a polymeric anion-exchange and/or cation-exchangeresin.

30. The method according to any of items 18-29, wherein theanion-exchange resin is AMBERLITE® IRA-400, AMBERLITE® IRA-485, DOWEX®1×2-100, DOWEX® 1-8-100, MARATHON® A2 or DIAION® SA 20A.

31. The method according to any of items 18-30, wherein the cationexchange resin is AMBERLITE® IRC-748, DOWEX® 50 WX2-100, DOWEX® 50WX8-100, MARATHON® MSC or DIAION® SK 110.

32. The method according to any of items 18-31, wherein the resin isnon-porous.

33. The method according to any of items 18-32, wherein the cell is aeukaryotic or prokaryotic cell.

34. The method according to any of items 18-33, wherein the cell isselected from Enterobacteriaceae, Pseudomonaceae, Lactobacteriacea, orBacillaceae.

35. The method according to item 34, wherein the cell is E. coli.

36. The method according to any of items 18-35, wherein the biomoleculeis a protein or a polynucleotide.

37. A method of disrupting cells comprising adding positively chargedand/or negatively charged microparticles or hydrophobic microparticlesto a cell suspension.

38. The method of item 37, further comprising releasing of biomoleculesfrom the cells.

39. The method of item 37 or 38, wherein the biomolecule is apolypeptide or polynucleotide.

40. A biological fluid comprising positively charged microparticlesand/or negatively charged microparticles or hydrophobic microparticles,wherein the positively charged microparticles comprise ground polymericanion-exchange resin, and wherein the negatively charged microparticlescomprise ground polymeric cation exchange resin.

41. The fluid of item 40 further comprising flocs.

42. Use of positively charged microparticles for biomolecule recovery,wherein the positively charged microparticles comprise ground polymericanion-exchange resin, and wherein the biomolecule is acidic or basic.

43. Use of item 42 for biomolecule recovery from cell lysate or cellhomogenate.

44. Use of item 42 for biomolecule recovery from cell suspension.

45. Use of negatively charged microparticles for biomolecule recovery,wherein the negatively charged microparticles comprise ground polymericcation exchange resin, and wherein the biomolecule is acidic or basic.

46. Use of item 45 for biomolecule recovery from cell lysate or cellhomogenate.

47. Use of item 45 for biomolecule recovery from cell suspension.

48. Use of positively and negatively charged microparticles forbiomolecule recovery, wherein the positively charged microparticlescomprise ground polymeric anion-exchange resin and negatively chargedmicroparticles comprise ground polymeric cation exchange resin, andwherein the biomolecule is acidic or basic.

49. Use of item 48 for biomolecule recovery from cell lysate or cellhomogenate.

50. Use of item 48 for biomolecule recovery from cell suspension.

51. Use of positively charged microparticles for cell disruption andrelease of biomolecule from the cell, wherein the positively chargedmicroparticles comprise ground polymeric anion-exchange resin, andwherein the biomolecule is acidic or basic.

52. Use of negatively charged microparticles for cell disruption,wherein the negatively charged microparticles comprise ground polymericcation exchange resin, and wherein the biomolecule is acidic or basic.

53. A method of obtaining biomolecules from a biological fluidcomprising a) adding positively charged microparticles and/or negativelycharged microparticles to a biological fluid, and recovering thebiomolecules from the biological fluid, wherein the biomolecule isacidic or basic.

54. The method of item 53 wherein the biological fluid is a cellsuspension, cell lysate or cell homogenate.

55. A method of obtaining biomolecules from a cell, comprising a) addingpositively charged microparticles and/or negatively chargedmicroparticles to disrupt the cell, thereby releasing the biomoleculefrom the cell, and b) recovering the released biomolecules.

56. The method of item 55, wherein the biomolecule is acid or basic.

57. The method of item 55, wherein positively charged microparticles isadded.

58. The method of item 55, wherein negatively charged microparticles isadded.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for biomolecule recovery and/orfor cell disruption that are simple and fast using the charged orhydrophobic microparticles as described herein. It is partly based onthe surprising finding that the microparticles, in particular thecharged microparticles can disrupt cells and can further release andadsorb biomolecules from the cells, thus enabling a fast and efficientrecovery of biomolecules. Moreover, it has been found that chargedmicroparticles rapidly form flocs of large diameters (such as at least 5μm) when added to a biological fluid such that the microparticles caninteract with/adsorb to cells and/or adsorb biomolecules, which allowsan easy separation of the biomolecules adsorbed to the microparticlesand/or flocks built by the microparticles and the biomolecules. The sameis true for hydrophobic microparticles.

As will be appreciated by a skilled person in the art, the presentinvention is particularly useful for separating proteins or plasmidsfrom biological fluids such as cell suspensions in large scaleapplications including pilot or industrial scale as described herein.The charged and/or hydrophobic microparticles can advantageously be usedin batch process (referred herein as “batch adsorption”).

Moreover, the novel methods described herein provides high recoveryefficiency and low contaminants levels. The present invention canadvantageously be applied for recovering biomolecules especially if thebiomolecules are temperature or shear sensitive and prone todenaturation when conventional methods of biomolecule extraction areapplied.

Cells and Biomolecules

As described earlier, the present invention provides methods forbiomolecule recovery from a fluid such as a biological fluid. In someembodiments, the methods of the present invention involve the disruptionof cells in the biological fluid and the release of biomolecules fromthe cells. The biomolecules are preferably intracellular biomoleculessuch as proteins or plasmids. The released biomolecules can besubsequently recovered from the biological fluid.

As defined herein, the term “intracellular” refers any substance foundwithin a cell. A “biomolecule” as defined herein includes any moleculethat are normally found in or synthesized by a cell, includingpolypeptides or polynucleotides. The biomolecules may be acidic or basicbiomolecules. Examples of biomolecules include but are not limited tooligosaccharide, polysaccharide, lipopolysaccharide, oligopeptides,proteins, nucleosides, flavonoids, oligonucleotides, DNA (ds or ssDNA),plasmid DNA, RNA (ds or ssRNA), organometallic compounds, amino acids,lipids, pyrimidines, purines, carbohydrates, peptidomimetic compounds,toxins, steroids and enzymes.

A “cell” when used herein refers to a cell, preferably a “host cell,”which is capable of producing (expressing) a biomolecule. Such cells canbe applied in the methods of the present invention. A foreign nucleotidesequence can be introduced in the cell for producing the biomolecule.

Cells or host cells that can be used in the method of the presentinvention can be either prokaryotic cells, eukaryotic cells, or both.More preferably, the cell used in the methods of the present inventionare vertebrate cells including mammalian, avian, amphibian and fishcells and insect cells. Also included by cells or host cells areeukaryotic cells. Typically, eukaryotic cells are mammalian cells, aviancells or insect cells. A cell or host cell also includes yeast cell orfungal cells. However, it is preferred that the host cell is aprokaryotic cell including bacterial cells from Gram-negative bacteriasuch as cells from Enterobacteriaceae, e.g. E. coli, orPseudomonadaceae, e.g. P. putida, or Gram-positive bacteria such ascells from Lactobacteriaceae or Bacillaceae. Most preferably however,the host cell is E. coli.

Microparticles

“Charged” microparticles as defined herein are positively chargedmicroparticles and/or negatively charged microparticles. “Positivelycharged” microparticles have at least one elementary charge of a proton,and more typically more than one, at a neutral pH. “Negatively charged”microparticles have at least one elementary charge of an electron, andmore typically more than one, at a neutral pH.

The microparticles can comprise ground resin. Resin useful for thepresent invention is a solid, non-soluble polymeric material which iscapable of interacting and attaching to various elements and allows forcapturing of the elements from a mixture. Resins are generally composedof inert compound including, but not limited to, sephadex, polystyrene,polyacrylamide, polymethacrylate or neutral polysaccharides. They mayalso include cross-linked natural polymers like cellulose, dextran oragarose.

Microparticles according to preferred embodiments are prepared fromion-exchange resin, more preferably, polymeric anion-exchange resinand/or cation-exchange resin. Ion exchange resin refers to a solidsupport containing insoluble carrier of an electrical charge polymerscarrying fixed functional groups or sites with exchangeable ions.Illustrative examples of suitable ion exchange resins for preparingmicroparticles include anion exchange resins, cation exchange resins,and mixed-mode chromatography resins, also sometimes referred to hereinas mixed-mode ion exchange resins. The exchangeable ion form isgenerally one or more of Na⁺, H⁺, OH⁻, or Cl⁻ ions, depending on thetype of ion exchangeable resin. Ion exchange resin includes weak andstrong acid cation exchange resins as well as weak and strong base anionexchange resins. Suitable ion exchange resins further include chelatingresins.

Ion exchange resins are widely used in various industrial fields. Ionexchange resins are commonly used, for example, in the field of watertreatment for demineralization of water for boilers or for condensatetreatment at power plants, in a food field for purifying a sugarsolution or in the field of super pure water for preparation ofsemiconductors.

In preferred embodiments of the present invention, microparticles areprepared from porous, spherical ion-exchange resins. Sphericalion-exchange resins are made by suspension polymerization, in which amonomer mixture comprising a monofunctional addition-polymerizablemonomer and a radical polymerization initiator are added to an aqueousmedium, followed by stirring to prepare a suspension of the monomermixture. The suspension is then maintained at a polymerizationtemperature for a period of time to obtain a spherical cross-linkedcopolymer. The diameter of ion-exchange resins for water treatment istypically between 300-600 In other embodiments, microparticles can beprepared from gel type and/or macroporous ion-exchange resins.

Polymer matrices of ion exchange resins may include polystyrene,polystyrene and styrene copolymers, polyacrylate, aromatic substitutedvinyl copolymers, polymethacrylate, phenol-formaldehyde, polyalkylamine,combinations thereof, and the like. In a preferred embodiment, thepolymer matrix is polystyrene and styrene copolymers, polyacrylate, orpolymethacrylate, and in another embodiment the polymer matrix isstyrene-divinylbenzene (DVB) copolymers. Preferably, the ion-exchangeresin for the preparation of microparticles uses resin which arepolystyrene-based, Hydroxyethyl methacrylate (HEMA)-based, dimethylaminoethyl methacrylate (DMAEMA)-based, dimethylamino ethyl methacrylate(pDMAEMA), methacrylic acid (MAA)-based. Most preferably, the resin ismade from polystyrene cross-linked with divinylbenzene (DVB).

The cation exchange resin used herein can be weakly or strongly acidic.As used herein, the term “weakly acidic cation exchange resin” refers toa resin having an apparent dissociation constant or ionization constant(pKa) greater than about 4.5 as measured by conventional methods (forexample, Fisher et al., “Effect of Cross-linking on the Properties ofCarboxylic Polymers. I. Apparent Dissociation Constants of Acrylic andMethacrylic Acid Polymers” J. Phys. Chem., 60(8), 1030 (1956)). It mayhave the carboxylic acid group, a phenolic hydroxyl group, a phosphonicacid group, and an arsono group as the exchange group. The term“strongly acidic cation exchange resin,” on the other hand, refers to aresin having a pKa less than about 1.5. A strongly acidic cationexchange resin may have sulfonic acid groups such as sodium polystyrenesulfonate or polyAMPS. The sulfonic acid group (—HSO₃) is the exchangegroup and behaves like a strong acid, dissociating to (—SO₃)⁻ and H⁺ inalkaline solutions and even in acidic solutions.

The anion-exchange resin used herein can be weakly or strongly basic. Asused herein, the term “weakly basic cation exchange resin” refers to aresin having an apparent dissociation constant or ionization constant(pKa) greater than about 8.5 as measured by conventional methods (forexample, Fisher et al., “Effect of Cross-linking on the Properties ofCarboxylic Polymers. I. Apparent Dissociation Constants of Acrylic andMethacrylic Acid Polymers” J. Phys. Chem., 60(8), 1030 (1956)). It mayhave the primary, secondary, and/or ternary amino groups, e.g.polyethylene amine as the exchange group. The term “strongly basic anionexchange resin,” on the other hand, refers to a resin having a pKa lessthan about 12. A strongly basic anion exchange resin may have quaternaryamino groups, for example, trimethylammonium groups, e.g. polyAPTAC, ordimethylethanolamine as the exchange group.

Anion-exchange resins and cation-exchange resins used herein furtherinclude chelating resins which comprise functional groups that arecapable of forming a chelate (complex) with a metal ion. Chelatingresins typically feature a great selectivity for specific metal ions.Some chelating resins can be basic and/or acidic, depending on theirfunctional groups and the pH. Typical functional groups of chelatingresins include, but are not limited to, iminodiacetic acid, polyamine,methylglucamide, thiouronium, and aminophosphonic acid. AMBERLITE® IRC748 is an exemplary chelating cation-exchange resin having a functionalgroup of iminodiacetic acid that can be used to prepare microparticlesfor the recovery of biomolecules from, e.g., a cell suspension.

Commercially available ion exchange resins are for example provided byRohm & Haas of Philadelphia, Pa., USA as AMBERLITE®, Amberjet, Duolite,and Imac resins, from Bayer of Leverkusen, Germany as Lewatit resin,from Dow Chemical of Midland, Mich. USA as Dow resin, from MitsubishiChemical of Tokyo, Japan as DIAION® and Relite resins, from Purolite ofBala Cynwyd, Pensylvania, USA as Purolite resin, from Sybron ofBirmingham, N.J. USA as lonac resin, and from Resintech of West Berlin,N.J. USA.

Positively charged microparticles can be prepared from polymeric anionexchange resin. Commercially available anion exchange resins aretypically in either OH⁻ or Cl⁻ forms. In one embodiment, the anionexchange resin is in the OH⁻ form. The resin may be for example“DIAION®” anion exchange resins such as DIAION® SA resins (includingDIAION® SA 20A) and DIAION® SK resins (including DIAION® SK 110) (fromMitsubishi Chemical) “AMBERLITE®” resins such as AMBERLITE® IRA-400,AMBERLITE® IRA-458, AMBERLITE® IRA-734, and AMBERLITE® IRA-900 (fromRohm & Haas Co.) or “DOWEX®” resins such as DOWEX® 1, DOWEX® 2, DOWEX®11, DOWEX® 21K, DOWEX® 1×2, DOWEX® 1×4, DOWEX® 1×8 and DOWEX® MARATHON®resins such as MARATHON® A2 (from Dow Chemical Co). In preferredembodiments, the anionic exchange resin is AMBERLITE® IRA-458 orMARATHON® A2. Functional groups in anion exchange resins may includequaternary ammonium groups, e.g., benzyltrimethylammonium groups (type 1resins), benzyldimethylethanolammonium groups (type 2 resins),trialkylbenzyl ammonium groups (type 1 resins), dimethylethanolamine(type 2 resins) or tertiary amine functional groups. For celldisruption, MARATHON® MA2 and AMBERLITE® IRA-458 are particularlypreferred anion exchange resins for the preparation of positivelycharged micro particles.

Negatively charged microparticles can be prepared from polymeric cationexchange resin. As used herein, a polymeric material may refer to apolymer, a mixture of polymers, a cross-linked polymer, mixturesthereof, or to polymeric networks. Often, a polymeric material is simplyreferred to as a polymer. Commercially available cation exchange resinsare typically in either H⁺ or Na⁺ forms. In one embodiment, a cationexchange resin is in the H⁺ form. The resin may be for example “DIAION®”cation exchange resins such as DIAION® PK resins and DIAION® SK resins(from Mitsubishi Chemical), “AMBERLITE®” resins such as AMBERLITE®IRC-748 (from Rohm & Haas Co.) or “DOWEX®” resins such as DOWEX® 50WX2,DOWEX® 50WX8, and DOWEX® MARATHON® resins such as MARATHON® C, MARATHON®MSC (from Dow Chemical Co). Functional groups of a cation exchange resinmay include sulfonic acid groups (—SO₃H), phosphonic acid groups(—PO₃H), phosphinic acid groups (—PO₂H), carboxylic acid groups (—COOHor —C(CH₃)—COOH), combinations thereof. In one embodiment, thefunctional groups in a cation exchange resin will be —SO₃H, —PO₃H, or—COOH, and in the most preferred embodiment, the functional groups in acation exchange resin is —SO₃H. For cell disruption, cation exchangeresins having a chelating functional group such as AMBERLITE® IRC 748are particularly preferred for the preparation of negatively chargedmicroparticles.

Polymeric cation exchange resin, as used herein, refers to a polymericmaterial having one or more elementary charges of the proton, or to sucha macromolecule itself. A polymeric anion exchange resin has one or moreelementary charges of the electron.

The positively charged microparticles of the invention are particleshaving at least one elementary charge of a proton, and more typicallymore than one, at a neutral pH, whereas the negatively chargedmicroparticles have at least one elementary charge of an electron at aneutral pH.

Positively or negatively charged microparticles are obtained when atleast a fraction of the constituents of the microparticles are ionicallycharged.

The present invention also provides as a novel adsorbent material forthe capture of biomolecules microparticles which are solid andhydrophobic. The microparticles are in ground form and can be preparedby grinding hydrophobic adsorbent material such as AMBERLITE® XAD4,AMBERLITE® XAD7HP, AMBERLITE® XAD761.

In one embodiment, only positively charged microparticles are added tothe biological fluid. In another embodiment, only negatively chargedmicroparticles are added to the biological fluid. Yet in anotherembodiment, both positively and negatively charged microparticles areadded to the biological fluid. If both positively and negatively chargedmicroparticles are added to the biological fluid, the ratio betweenpositively charged microparticles and negatively charged microparticlescan be from about 0.1:99.9 (w/w) to 99.9:0.1 (w/w). For example, it canbe about 50:50, but it can also be different, such as 90:10, 80:20,75:25, 60:40, 40:60, 20:80, 25:75, 10:90, etc. In yet anotherembodiment, hydrophobic microparticles are added to the biologicalfluid.

In one preferred embodiment, the microparticles are in the form ofground particles having an average particle size less than about 10 suchas less than about 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, and 1μm. Preferably, the ground particles have an average particle size lessthan about 5 μm, and more preferably less than 2.5 μm. Preferably, theground particles have an average particle size larger than 0.5 μm.Accordingly, the ground particles may preferably have an averageparticle size in the range from about 10 μm to 0.5 μm, about 9 μm toabout 0.5 μm, about 8 μm to about 0.5 μm, about 7 μm to about 0.5 μm,about 6 μm to about 0.5 μm, about 5 μm to about 0.5 μm, about 4 μm toabout 0.5 μm, about 3 μm to about 0.5 μm, or about 2.5 μm to about 0.5μm. However, the ground particles may have a particle size more than 10μm as well as less than 0.5 μm.

Preparation of Microparticles

Microparticles are obtainable or obtained by grinding anion-exchangeresin and/or cation exchange resin. Preferably, the microparticles ofthe present invention are obtainable by (or are obtained by) grindingthe resin and conditioning the resin.

It is preferable to condition the ground particles to remove residualby-products in the manufacturing process of the resin. Typicalconditioning methods for ion exchange resins are well known in the artand also described by the suppliers.

If necessary, “conditioning” can be performed in order to transfer theresin from the H⁺ or OH⁻ to Na⁺ or Cl⁻ form. In one embodiment,conditioning is performed by repeated washing steps using NaCl andwater. In the process, the resin can be ground in water andsedimentation can be done by centrifugation. Alternatively, resinsalready in Na⁺ or Cl⁻ form are available commercially and can beobtained from the suppliers.

In a preferred embodiment the microparticles are prepared by (a)grinding the ion exchange resin and (b) resuspending said ground resinin water, (c) allowing sedimentation of said ground resin, (d)collecting ground resin from the supernatant of the sedimentedsuspension, (e) resuspending collected ground resin in about 2 M sodiumchloride, (f) allowing sedimentation of said ground resin, (g)collecting ground resin from the supernatant of the sedimentedsuspension of (f), (h) allowing sedimentation of said ground resin, (i)collecting the sediment of the ground resin of (h), and (j) washing saidcollected ground resin.

Hydrophobic microparticles of the present invention are preferablygrinded overnight Grinded resins are suspended in water. Supernatant iscentrifuged. Resins are re-suspended in salt solution, such as 2 Msodium chloride and centrifuged, a pellet is discarded. Supernatant aretransferred and centrifuged again. Supernatant is discarded. Groundresins are re-suspended in water and transferred to tubes. Resins arecentrifuged, supernatant is discarded and resin is re-suspended inaqueous washing solution. Wash sequence is:

-   -   1×50% EtOH (dilution of organic residues)    -   3× deionized water (dilution of EtOH)        Grinding

Grinding can be carried out in any way known in the art, including, butnot limited to, by a grinding device, such as a grinding mill (includinga jet mill, a ball mill, a hammer mill or the like), or by hand with forexample a mortar and pestle. “Grinding” as used herein refers to anoperation leading to a reduction in the particle size. A skilled personcan readily select grinding methods to prepare the resins. For example,in one embodiment, the resin is wet ground in an automated manner bymoving one or more pestles in a mortar. The grinding process may becontinued until the majority of the particles have a size of less thanabout 10 μm, such as less than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 μm, areobtained. By majority it is meant more than 50%, such as more than 60%,70%, 80%, 90%, or 95%. In other embodiments, the majority of theparticles have an average particle size of at least 0.1 μm, such as 0.2μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1μm, 1.2 μm, 1.3 μm, 1.4 μm 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0μm, 3 μm, 4 μm or 5 μm.

A skilled person can readily determine the size of ground particles withmethods known in the art. From that, the average particle size can bedetermined by means and methods known in the art. For example, the sizecan be determined by optical microscopy using a software-baseddetermination of size such as illustrated in the example. Particle sizeof ground resin can be determined at 1000-fold magnification byestimation of equivalent circular diameters. Distribution is preferablycalculated by comparison of diameter sizes of about 100-500 particles at1% v/v. Grinding has the effect of drastically increasing surface area,which leads to a significant increase in the binding capacity ofbiomolecules particularly for proteins or polypeptides. Determination ofthe diameter is preferably done with the aid of technical means such asa software which recognizes a particle and measures the diameter.

“Resuspending” or “suspending” or any grammatical form thereof when usedherein means that microparticles are brought into suspension.

“Allowing sedimentation” when used herein means that microparticles areallowed to settle out of the fluid in which they are entrained and cometo rest against a barrier. The sedimentation is due to the particles'motion through the fluid in response to forces acting on them. Theseforces can be gravity or centrifugal acceleration by, e.g., acentrifuge, with the latter being preferred.

“Collecting” means that microparticles are harvested from thesuspension.

“Washing” when used herein means that residual amounts of fluids thatcould disturb or interfere with the performance of the microparticlesare reduced. For example, the resins can be washed with 50% (v/v)ethanol (EtOH) and double-deionized water (ddH₂O), preconditioned asdescribed above, and subsequently washed repeatedly with ddH₂O. Thevolume of each of these fluids is in excess of the volume ofmicroparticles, preferably 10- or 20-fold in excess.

After the grinding process, particles outside the preferred range can beoptionally removed, for example, by centrifugation, sedimentation,filtration, or any other methods known to a skilled person in the art.

Surprisingly, it has been found that the rough surface in the groundparticles provides a comparable adsorption capacity for proteins likeconventional chromatographic media having high binding capacity such asthe Nuvia media developed by Bio-Rad Laboratories (USA).

Addition of the Microparticles

In the first step, the microparticles are added to the biological fluid.The presently disclosed microparticles can be used in laboratory scale,pilot-scale or industrial scale. As used herein, “laboratory scale”comprises batch adsorption of a biomolecule from about 1 or 10 ml fluidto about 1000 ml fluid. As used herein, “pilot-scale” comprises batchadsorption of a biomolecule from about 1 liter fluid to about 10 literfluid. As used herein, “industrial scale” or large-scale comprises batchadsorption of a biomolecule from about 10 liter fluid to about 1000 oreven 10000 or more liter fluid.

The method described herein comprises adding positively chargedmicroparticles, or adding negatively charged microparticles or addingboth positively and negatively charged microparticles or hydrophobicmicroparticles into the biological fluid. If both positively andnegatively charged microparticles are added, they can be added as aprepared mixture, or separately, in a simultaneous or successivefashion. If positively and negatively charged microparticles are addedsuccessively, i.e. one after another, the present invention thusencompasses first adding the either positively charged microparticles ornegatively charged microparticles to the biological fluid, and secondlyadding the oppositely charged microparticles to the biological fluid. Askilled person will be able to determine whether only positively chargedmicroparticles or only negatively charged microparticles or bothpositively and negatively charged microparticles are used consideringfor example the biological fluid and the desired biomolecule to berecovered from the biological fluid.

In the alternative, the adsorbent according to the present inventioncomprises hydrophobic resin in the form of ground particles. Theoutstanding protein adsorption capacities of such hydrophobicmicroparticles, which are superior to conventional chromatographic mediaat low salt concentration, are especially useful for e.g. negativepurification of polynucleotides or hydrophilic proteins. Accordingly,the present invention provides uses and methods for negativepurification of polynucleotides or hydrophilic protein by applying thehydrophobic microparticles. For that purpose To a homogenate or standardprotein solution, 50% (v/v) hydrophobic microparticles suspensions areadded to homogenate or protein solutions. Microparticles suspensions areincubated, e.g. for 30 minutes. Afterwards microparticles arecentrifuged and elution of bound protein is performed by addition ofelution buffer, mixing and incubation for e.g. 30 min. Optionally, asecond washing step with elution buffer can be included. After elutionmicroparticles are centrifuged again as before. Concentration of proteinin supernatants can be quantified by e.g. photometric analysis andpurity of target protein can be checked by SDS-PAGE.

The microparticles can be added into the biological fluid from whichbiomolecules are to be separated. The term “biological fluids” should beunderstood broadly. They refer to any fluid associated with organisms,such as obtained from or produced by any organisms. Examples ofbiological fluids include cell culture media, fermentation supernatants,fermentation broths, cell suspensions, cell lysate. Further examples ofbiological fluids are described herein above. In other embodiments,biological fluids may also be saliva, urine, lymphatic fluid, prostaticfluid, seminal fluid, blood, plasma, sera, sweat, mucous secretion,milk, milk whey, ascites fluid, organ extracts, plant extracts, animalextract. In a preferred embodiment, the biological fluid is anybiological fluid described herein, such as a polypeptide orpolynucleotide, e.g., plasmid DNA, cosmid DNA, BAC DNA, minicircle DNA,etc. containing fluid, derived from various in vitro or in vivoprocesses, and particularly, fermentation broth, culture broth,fermentation supernatant, culture supernatant, cell homogenate, celllysate, or cell suspension. “Cell homogenate” is generally understood asa mixture of broken cells. Cell homogenate may be obtained by amechanical or chemical method. For example, cells can be homogenized byconventional methods such as high pressure in a homogenizer to render afermentation homogenate, or by simply mixing in a lysis solution,including alkaline lysis.

Therefore, the present invention also includes a fluid comprisingbiomolecules and positively and negatively charged microparticles. Inpreferred embodiments, the biological fluid is agitated during and/orafter any of the steps of the methods of the present invention, butpreferably not during the step when the particles are allowed to formflocs and/or when the flocs are removed from the biological fluid.

“Cell homogenate” is generally understood as a mixture of broken cells.Cell homogenate may be obtained by a mechanical or chemical method. Forexample, cells can be homogenized by conventional methods such as highpressure in a homogenizer to render a cell homogenate/lysate, or bysimply vortexing the cells in a lysis solution, including alkalinelysis.

Examples of biological fluids include cell cultures, cell homogenates,and cell lysates and fermentation supernatants such as from E. coli, andCHO cell culture. The fermentation supernatants or cell homogenates canadditionally be filtered, concentrated, dialyzed, conditioned, ortreated in another way.

In a preferred embodiment of the present invention, the biological fluidis a cell suspension. The term “cell suspension” as used herein refersto a liquid, such as cell culture medium, buffer, or any other suitableliquid, that comprises preferably intact cells. “Intact” refers to thephysical continuity of the cellular membrane enclosing the intracellularcomponents of the cell and means that the cellular membrane has not beendisrupted in any manner that would release the intracellular componentsof the cell to an extent that exceeds the permeability of the cellularmembrane under conventional culture conditions.

During and/or after the microparticles are added into the biologicalfluid, they can be mixed by stirring or shaking (only after the additionof the MPs) to obtain a homogenous mixture. In some embodiments, mixingcan enhance flocculation and/or cell disruption by facilitating thecontact between cells and/or biomolecules and microparticles. Adsorptiontakes place spontaneously while the particles are mixed with thebiological fluid. However, in some embodiments, no mixing is requiredafter the addition of the microparticles (referred as static incubationin the examples).

Incubation Parameters

When microparticles are added to biological fluid, the optimalvolumetric concentration (indicated by “% (v/v)” which refers to theratio of the respective volumetric fractions) of cells may be adjusted.In some embodiments, the volumetric cell concentration is less than 30(v/v), such as less than about 25% (v/v), less than about 20% (v/v),less than about 15 (v/v), less than about 10% (v/v), less than about 9%(v/v), less than about 8% (v/v), less than about 7% (v/v), less thanabout 6% (v/v), less than about 5% (v/v), less than about 4 (v/v), lessthan about 3% (v/v), less than about 2% (v/v) or less than about 1%(v/v). Mixing may be useful in order to obtain a homogenous mixture ofcells and microparticles.

In some aspects of the present invention, the microparticleconcentration is preferably less than about 300% (v/v), such as lessthan about 200%, 100% (v/v), 80% (v/v), 70 (v/v), 60% (v/v), 50% (v/v),40% (v/v), 30% (v/v), 20% (v/v), 10% (v/v) or less. Selection ofvolumetric ratios of resin:cells can depend on, for example,microparticle size distributions (effective surface area) and chargedensities (functional groups per accessible area).

Flocculation

In some embodiments, the next step after addition of the microparticlesto the biological fluid is to allow the formation of flocs. It has beensurprisingly found that the microparticles can rapidly form flocs oflarge diameter with the cells and/or the biomolecules in the biologicalfluid upon adsorption of the cells and/or the biomolecules to themicroparticles.

When the microparticles are added to a cell suspension, cells may beimmobilized in flocs by adsorption to the microparticles. In someembodiments, the cells release biomolecules and remain viable. Thereleased biomolecules may or may not adsorb to the micro particles.

When the microparticles are added to a biological fluid, such as a celllysate or a cell homogenate or a fermentation supernatant, flocs mayform upon adsorption of biomolecules to the microparticles. In someembodiments, positively and negatively charged microparticles are firstmixed and then added to a biological fluid, and flocculation occurs uponcontact of the microparticle mixture with the biological fluid. In otherembodiments, first positively or negatively charged microparticles areadded to the biological fluid, and subsequently the oppositely chargedmicroparticles are added separately, resulting in the formation offlocs.

In one embodiment the biomolecule is acidic. In this case, positivelycharged microparticles is added to a biological fluid such as a celllysate or cell homogenate for adsorption. Positively chargedmicroparticles may also be added to a cell suspension, either at anamount only sufficient to disrupt the cell and to release thebiomolecule, or at an higher amount which will disrupt the cell as wellas adsorb the biomolecules. A skilled person is able to determine theamount necessary to partially or fully disrupt the cell. Negativelycharged microparticles may be added thereafter, which works ascross-linker to increase the particle size and stability of the flocs.Alternatively, negatively charged microparticles such as prepared fromchelating cation exchange resin may also be added to the cell suspensionat an amount sufficient to disrupt the cell and to release thebiomolecule for further purification. Optionally, positively chargedmicroparticles may be afterwards added to increase flocculation.

In another embodiment the biomolecule is basic. In this case, positivelycharged microparticles may added to a biological fluid such as a celllysate or cell homogenate to form flocs with the cell debris or otherimpurities such as DNA, host cell proteins and cell fragments.Negatively charged microparticles may be added to increase the particlesize and stability of the flocs, so the flocs can be easily separatedand discarded. The basic biomolecules can then be recovered from thesupernatant because it would not bind to the positively chargedmicroparticles. Alternatively, positively or negatively chargedmicroparticles may be added to a cell suspension at an amount sufficientto disrupt the cell and to release the biomolecule for furtherpurification. The flocs typically have a size of 100 μm or even largerwhich makes them visible and facilitates their separation. This meansthat other unwanted material such as cells and/or cell debris can beeasily removed by filtration, rendering centrifugation unnecessary. Thepresent invention is therefore faster and simpler than prior artmethods. It is not necessary but possible to regenerate the resin whichwould otherwise be required if column chromatography was used.Furthermore, the microparticles are a cheap material and thus can bediscarded after use.

In preferred embodiments, flocs have an average particle size of atleast 5 μm, such as at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 μm, 2000 μm or more areformed.

Adsorption Capacity

The “adsorption capacity” as used herein is defined as the quantity ofadsorbed biomolecule (in mg) per ml of resin in equilibrium state.Equilibrium as used herein is the state wherein the rate of adsorptionequals the rate of desorption. The adsorption capacity of themicroparticles for the desired biomolecule, in particular a solublepolypeptide or polynucleotide, can for instance be determined byquantifying said polypeptide or polynucleotide in the supernatant, forexample by fluorescence or spectrophotometry, before and after elutionof the biomolecules. The difference of the biomolecule quantity is thenconsidered to be adsorbed to the microparticles. As the person skilledin the art will understand, the adsorption capacity can depend onvarious parameters, such as characteristics of the microparticles, thebiomolecules, pH, temperature, salt concentration, and other parameters,or combinations thereof. In some embodiments, positively chargedmicroparticles can adsorb at least 5 mg, such as at least 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 mg GFP per ml resin under the conditions as set forth in Example3.1.5. In other embodiments, positively charged microparticles canadsorb at least 5 mg, such as at least 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg SOD per mlresin under the conditions as set forth in Example 4.1.3.

Removal of Flocs

Generally, removal of the flocs from a liquid (such as a biologicalfluid or a buffer) can be performed by filtration, centrifugation,sedimentation, or any other suitable means. A skilled person can readilydetermine what methods can be used to separate or desorb the flocs fromthe fluid. The suspension of the flocs can for instance be processed ineither a bucket centrifuge (laboratory scale), tubular centrifuge,decanter or disk stack centrifuge for pilot and industrial scaleoperation. Likewise, it is possible to remove the flocs by filtrationwhere the flocs are retained, or by sedimentation or extraction.Desorption can be achieved by counter-current extraction decanter,mixer-settler or column extractor. Other useful methods for removal maybe tangential flow filtration, deep-bed filtration, Dead End Filtration,or methods involving the use of filter press, nutsche filter.

Desorption of Biomolecules

Desorption can be carried out using any methods known in the art. Forexample, desorption can be carried out by resuspending the flocs in abuffer which allows desorption of biomolecules like proteins (desorptionbuffer). This can be achieved by using any known means in the art,including a tubular (static) mixer or other mixing devices such asstirred-tank. Desorption can also be achieved by counter-currentextraction decanter, mixer-settler or column extractor.

The suspension is then subject to conditions suitable for desorption. Askilled person can readily determine such conditions for desorbing thebiomolecules adsorbed on the flocs. Generally, desorption methods usedin conventional ion-exchange chromatography can be employed. Forinstance, desorption can be carried out by elution at a pH below orabove the isoelectric point or by increased salt concentration.

The biomolecules can be further purified or enriched by methods known inthe art. These include, for example, precipitation, crystallizationand/or chromatography selected from the group consisting of hydrophobicinteraction chromatography, affinity chromatography, pseudo-affinitychromatography, anion or cation exchange chromatography and/or sizeexclusion chromatography. Accordingly, the methods described hereininclude in a preferred embodiment a further step of purifying and/orenriching the desired biomolecule, in particular a protein, by makinguse of precipitation and/or chromatography.

However, it has been discovered that in some embodiments, the cells canrelease biomolecules upon interaction with said microparticles withoutadsorbing to the microparticles. In this case, cells do not adsorb tothe microparticles and no flocculation will occur. For example, whencharged microparticles are added to a cell suspension of cells carryingan opposite surface net charge, the cells do not adsorb to themicroparticles and no flocculation will be observed. The “surface netcharge” of a cell is herein defined as the sum of all electric chargespresent at the cellular surface which may be dependent on the pH of thesurrounding solution.

The released biomolecules may or may not adsorb to the microparticles.If the biomolecules do not adsorb to the microparticles, no desorptionof biomolecules is carried out. In this case, the biomolecule can berecovered from the supernatant by separating cells and microparticlesfrom the fluid, e.g. by centrifugation or filtration or any other means.If the biomolecules adsorb to the microparticles, desorption is carriedout by altering the condition of the microparticles to allow the elutionof the biomolecules for example by a desorption buffer. Due to the sizeof the flocs, after the biomolecules are desorbed by a desorptionbuffer, it is easy to separate the flocs formed from the desorptionbuffer.

The person skilled in the art readily knows which methods to apply inorder to achieve separation of supernatant from microparticles and/orcells.

Recovery

The present invention can be used to recover biomolecules from abiological fluid and/or cell. Recovering the biomolecule in all itsgrammatical forms can mean that a biomolecule is obtained, harvested,achieved, received or gained. The biomolecules may be plasmids,polynucleotides or expression products such as peptide, proteins,including proteins that are glycosylated or post-transnationallymodified. By means and methods known in the art and/or described herein,the biomolecule may be isolated and/or further processed such as furtherpurified. Moreover, recovery also includes the embodiment that the cellsare disrupted to release the biomolecules from the cell, rendering itsseparation from the cell culture possible. As subsequent steps, furtherpurification and/or enrichment of the biomolecule can be carried out.

Cell Disruption

The present invention further provides a method for cell disruption byadding charged microparticles to a cell suspension. The term “celldisruption” or “disruption of cells” are used interchangeably herein fora method or process for making a cell permeable to such an extent thatbiomolecules are released from the cell. Cell disruption may or may notinvolve cell death. Preferably, cell disruption does not involvecomplete fragmentation of cellular structures, such as the cell wall,resulting in a decrease of cell fragmentation which reduces the level ofunwanted contamination including cell debris. In some embodiments of theinvention, cells that are disrupted by the method described hereinrelease biomolecules and remain viable. The term “viable” refers tocells which are capable of multiplying under suitable growth conditions.

The released biomolecules may or may not adsorb to the microparticlesused to disrupt cells. The biomolecules can subsequently be recoveredfrom the biological fluid using the methods described herein or otherknown techniques. Thus, the present invention offers a novel method forcell disruption, biomolecule release and subsequent biomolecule recoveryin a simplified two-step process. Microparticles of the presentinvention may be used to open up the cell to release biomolecules sothat they could be recovered in the cell suspension, irrespective of theacidity of the biomolecules (i.e. the biomolecule may be acidic, basicor neutral).

Selectivity

The methods of the present invention can provide biomolecules of ahigher purity compared to biomolecules obtained by conventional methodsof cell disruption. In particular, the methods of the present inventionhave a higher selectivity than conventional methods used in the priorart due to a lower amount of non-target substances in the recoveredbiomolecule fraction. A high purity of the recovered biomolecule isfavourable because it can obviate the need for further successivepurification steps. Preferably, the present methods provide biomoleculeswith a relative enrichment to non-target biomolecules of more than 30%,such as more than 40%, more than 50%, more than 60%, more than 70%, morethan 80%, more than 90%, or even 100%. Methods for assessing the purityof a given biomolecule in comparison to non-target biomolecules areavailable to the person skilled in the art. For polypeptides, anexemplary method is the quantitative densitometry of proteins stainedwith Coomassie blue after separation on SDS polyacrylamidegelelectrophoresis (SDS-Page).

When conventional methods for cell disruption are applied, cells arebroken and nucleic acids, cell wall components and other fragments arereleased. Thus, the recovered biomolecules have to be further purifiedin order to remove the contaminants. However, the methods describedherein enable a reduced release of macromolecular contaminants. Suchcontaminants may include, but are not limited to, dsDNA, RNA, host cellproteins, host cell debris and endotoxins. It has been surprisinglyfound that the methods of the present invention markedly reduce thedsDNA content up to 2, 3, 4, 5, 6, 7, 8, 9 or 10 times or even morecompared to the dsDNA content in a cell homogenate obtained by astandard HPH protocol as described in Example 2.2. The person skilled inthe art can determine the dsDNA content in a given sample, for exampleby fluorimetric or colorimetric assays that are commercially available.Preferably, 5 times less dsDNA is released by the cells when applyingthe methods of the present invention, and more preferably 10 times lessdsDNA, or even less, such as 100 times, or 1000 times less dsDNA isreleased.

“Endotoxin” as used herein is used interchangeably withlipopolysaccharide (LPS), which is a major constituent of the outer cellmembrane of Gram-negative bacteria. Endotoxin is typically released upondestruction of the bacterial outer cell membrane. The methods of thepresent invention have been surprisingly shown to reduce the amount ofreleased endotoxin compared to the endotoxin content in a cellhomogenate obtained by a standard HPH protocol as in Example 2.2.Preferably, 5 times less endotoxin, more preferably 10 times lessendotoxin, or even less, such as 100 times, 1000 times, 10⁴ times, 10⁵or 10⁶ times less endotoxin, is released by the cells when applying themethods of the present invention. The person skilled in the art canreadily determine the endotoxin content for example by using the LimulusAmebocyte Lysate (LAL) gel clot test, LAL chromogenic tests and otherchromogenic tests that are commercially available or known in the art.

Cultivating Cells which Produce Biomolecules (“the Product”)

Prior to applying the adsorbent of the invention, the method ofobtaining a biomolecule as defined and described herein may optionallycomprise the step of cultivating a (host) cell that produces, such asexpresses, a biomolecule (the “product”), preferably an expressionproduct such as a protein or polynucleotide. The term “cultivation ofcells” or “culturing of cells” in medium (either with serum or serumfree) in the context of the host cells of the present invention refersto the seeding of the cells into the culture vessel, to the growing ofthe cells in medium until, in case of adherent culturing, a monolayer isformed, or, in case of a suspension culture, a sufficient cell densityis established and/or to the maintenance of the cells in medium as soonas the monolayer is formed or to the maintenance of the cells insuspension, respectively. The term “cultivation of cells” or “culturingof cells” in medium also includes that all of the above mentioned stepsare performed with serum free medium, so that no or essentially noanimal serum products are present during the whole cultivation processof the cells. Cells may be cultivated by exponential feed, or linear orconstant feed or other type of feed, fed batch cultivation, or highdensity cultivation. Yet, in the alternative, the above mentioned stepsmay also be performed with serum containing medium.

The nucleotide sequence and/or the encoded polypeptide may or may not beheterologous with respect to the cell. By “heterologous,” this meansderived from a cell or organism with a different genomic background, oris homologous with respect to the (host) cell, but located in adifferent genomic environment than the naturally occurring counterpartof said nucleotide sequence. This means that, if the nucleotide sequenceis homologous with respect to the host, it is not located in its naturallocation in the genome of said host, in particular it is surrounded bydifferent genes.

In a preferred embodiment of the present invention, the expressionproduct is a proteinaceous product. “Proteinaceous” when used hereinrefers to any of a group of complex organic macromolecules that containcarbon, hydrogen, oxygen, nitrogen, and usually sulphur and are composedof one or more chains of amino acids. A preferred proteinaceousexpression product is a polypeptide. The term “proteinaceous” also meansrelating to, consisting of, resembling, or pertaining to protein. In amore preferred embodiment of the present invention, the product is apolypeptide of interest which is produced. It is preferred that theproduct is biologically active. The proteinaceous product may be acidicor basic.

The expression product can be the product of transcription and/ortranslation of a nucleotide sequence, preferably of a nucleotidesequence that is exogenously added to the cell by means and methodscommonly known in the art in the context of genetically engineering hostcells. The product can be a nucleotide sequence including, for example,a plasmid, mini-circle DNA, cosmid, BAC, ssDNA or dsDNA sequence or RNAsequence (ribozyme, antisense RNA, sRNA, iRNA, miRNA and the like), allof which are capable of being produced in the cell, or it can be apeptide or polypeptide that is generated by way of translation of thetranscribed RNA in the cell.

A “polypeptide” as used herein includes proteins, peptides, polypeptidesand fragments thereof, said “polypeptides” all being preferablybiologically active. The terms “polypeptide” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length,generally more than about 10, 20 or 30 amino acids. These terms alsoinclude proteins that are post-translationally modified throughreactions that include glycosylation, acetylation and phosphorylation.The polypeptide may be a fusion polypeptide fused to a fusion partnerfor half-life extension, such as Fc-fusions, albumin-fusions, or fusionpartners as affinity tag for affinity chromatography, or fusion partnersfor providing correct N-termini or for increasing production yield ofthe protein of interest. The term “peptide” refers to shorter stretchesof amino acids, generally less than about 30 amino acids. A polypeptidecan serve as agonist or antagonist, and/or have therapeutic ordiagnostic uses.

Further, a polypeptide expressed in a cell of the present invention canbe of mammalian origin although microbial and yeast products can also beproduced.

Examples of mammalian polypeptides or proteins include hormones,cytokines and lymphokines, antibodies such as Fabs, nanobodies, dAbs,scFvs, receptors, adhesion molecules, and enzymes as well as fragmentsthereof. A non-exhaustive list of desired products include, e. g., humangrowth hormone, bovine growth hormone, parathyroid hormone, thyroidstimulating hormone, follicle stimulating hormone growth, luteinizinghormone; hormone releasing factor; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; calcitonin; glucagon;molecules such as renin; clotting factors such as factor VIIIC, factorIX, tissue factor, and von Willebrands factor; anti-clotting factorssuch as Protein C, atrial natriuretic factor, lung surfactant; aplasminogen activator, such as urokinase or human urine or tissue-typeplasminogen activator (t-PA); bombesin; thrombin; hemopoietic growthfactor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES(regulated on activation normally T-cell expressed and secreted); humanmacrophage inflammatory protein (MIP-1-alpha); a serum albumin such ashuman serum albumin; mullerian-inhibiting substance; relaxin A- orB-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase;inhibin; activin; receptors for hormones or growth factors; integrin;protein A or D; rheumatoid factors; a neurotrophic factor such asbone-derived neurotrophic factor (BDNF), neurotrophin-3,-4,-5, or -6(NT-3, NT-4, NT-5, or NT-6), growth factors including vascularendothelial growth factor (VEGF), nerve growth factor such as NGF-;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF, bFGF, FGF-4, FGF-5, FGF-6; epidermal growth factor (EGF);transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-p1, TGF-p2, TGF-p3, TGF-p4, or TGF-p5; insulin-like growthfactor-I and -II (IGF-I and IGF-11); des (1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD-3,CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (Ls), e.g., IL-1 to IL-10;superoxide dismutase; erythropoietin; T-cell receptors; surface membraneproteins e.g., HER2; decoy accelerating factor; viral antigen such as,for example, a portion of the AIDS envelope; transport proteins; homingreceptors; addressins; regulatory proteins; antibodies; chimericproteins such as immunoadhesins and fragments of any of the above-listedpolypeptides.

Preferred polypeptides and proteins herein are therapeutic proteins suchas TGF-β, TGF-α, PDGF, EGF, FGF, IGF-I, DNase, plasminogen activatorssuch as t-PA, clotting factors such as tissue factor and factor VIII,hormones such as relaxin and insulin, cytokines such as IFN-y, chimericproteins such as TNF receptor IgG immunoadhesin (TNFr-IgG) or antibodiessuch as bispecific antibodies, camelid antibodies and fragments thereof,V_(HH) domain antibodies, domain antibodies, immunoglobulins such asanti-IgG, anti-IgA, anti-IgM, anti-IgD or anti-IgE. Preferredtherapeutic proteins are those of human origin or “humanized” proteinssuch as humanized antibodies as described herein.

If the product is a polypeptide, the polypeptide can be tagged, i.e.,fused with a heterologous polypeptide which preferably allows isolationand/or purification of said polypeptide. The heterologous polypeptidecan, for example, be a histidine tag, Flag-tag, streptavidin tag, strepII tag, an intein, maltose-binding protein, an IgA or IgG Fc portion,protein A or protein G.

If the product is a polynucleotide including a nucleotide sequence, thenucleotide sequence may be fused with a heterologous nucleotide sequencewhich allows isolation and/or purification of said expression productbeing a nucleotide sequence. For example, the heterologous nucleotidesequence can bind to a complementary nucleotide sequence, therebyallowing isolation and/or purification of said nucleotide sequence.“Heterologous” when used in the context of a heterologous polypeptide ornucleotide sequence means that a polypeptide or nucleotide sequence isdifferent from the polypeptide or nucleotide sequence being the desiredexpression product.

If the product, as an example of a polynucleotide, is a plasmid, saidplasmid is useful for gene therapy or DNA vaccination, or may encode atherapeutic protein, such as one described herein.

On the other hand, the cell may express a virus, i.e., the host cellserves as producer cell line that provides, so to say, the appropriateenvironment that the virus replicates and/or is propagated. Accordingly,the product could be a virus. Virtually, any virus can be recovered bythe methods of the present invention such as dsDNA viruses (e.g.Adenoviruses, Herpesviruses, Poxviruses), ssDNA viruses (e.g.Parvoviruses), dsRNA viruses (e.g. Reoviruses), (+) ssRNA viruses (e.g.Picornaviruses, Togaviruses), (−) ssRNA (e.g. Orthomyxoviruses,Rhabdoviruses), ssRNA-RT viruses (e.g. Retroviruses) and dsDNA-RTviruses (e.g. Hepadnaviruses). Viral replication is the term used todescribe the formation of virus during the infection and propagationprocess in the target cells. From the perspective of the virus, thepurpose of viral replication is to allow production and survival of itskind. By generating abundant copies of its genome and packaging thesecopies into viruses, the virus is able to continue infecting new hosts.In the context of the present invention it is preferred that virusesproduced by appropriate host cells are not or essentially not capable ofexiting the host cell, for example, by way of lysis or budding.

As mentioned before, the product may also be a virus. A “virus” includes“native” viruses and “recombinant” viruses, with “native” meaning avirus which is isolated from nature and not genetically engineered (suchas a clinical isolate) or a virus which can be found in nature (i.e.,naturally-occurring) or a typical, established virus strain, for exampleused for immunization purposes (such as an attenuated virus).

In sum, the present invention provides a fast, efficient, simplified andinexpensive method which can be easily applied on an industrial scale.

EXAMPLES Example 1: Preparation of Micro Particles from Ion ExchangeResins

Resin beads for water treatment were purchased from DOW. An overview oftested resins is shown in Table 1.

TABLE 1 Resin Function Matrix Exchanged Name Polymer Type Group Type ionCapacity MARATHON ® Styrene- Macroporous Sulfonic acid Strong Cation≥1.7 (Na) MSC DVB acid MARATHON ® Styrene- Gel Dimethylethanol StrongAnion ≥1.2 (Cl) A2 DVB ammonium base AMBLERLITE ® Styrene- MacroporousImminodiacetic Chelating Cation ≥1.25 IRC748 DVB acid (Na) AMBLERLITE ®Acrylic Gel Trimethyl Strong Anion ≥1.25 IRA458 ammonium base (Cl)

All resins were preconditioned to their sodium or chloride form forcation exchangers (MARATHON® MSC, AMBERLITE® IRC748) and anionexchangers (MARATHON® A2, AMBERLITE® IRA458), respectively. Afterwardsparticles were repeatedly washed with deionized water (<1 mS/cm and pHneutral). An overview of functional groups of resins is showed in FIG.1.

Microparticles were wet ground at the company NETSCH with a Labstar LS1mill or manually with a pestle and mortar. An overview of averageparticle size distribution (PSD) obtained by grinding is shown in Table2. Particle sizes were measured by optical microscopy at 1000×magnification and average equivalent circular diameter was estimated bycounting 500-50000 discrete projections.

TABLE 2 Particle size distributions Size d50 Distribution d1-d99 Resin(μm) (μm) MARATHON ® MSC 0.9 0.4-1.9 MARATHON ® A2 1 0.4-2.4 AMBERLITE ®IRC748 1 0.2-4.5 AMBERLITE ® IRA458 1.4 0.4-5.1

Shapes of ground particles from optical projections were consideredundefined. Suspensions concentration was adjusted by centrifugation andestimated from packed bed volume in deionized water. Water content wascalculated from weight difference of wet and dried resin.

Example 2: Recovery of Target Proteins Example 2.1 Recovery of TargetProteins by CSPE

E. coli (HMS174) cells were cultivated in fed batch at 37° C.(GFPmut3.1) and 30° C. (SOD). Expression of recombinant protein was IPTGinduced. Cells were collected by centrifugation. Protein extractionexperiments were performed with aliquots at room temperature. Initiallyvolumetric cell concentration (wet packed bed) was approximately 10%v/v. This was determined by centrifugation of 50 ml suspensions at 4000rcf for 10 min. For experiments with various cell and saltconcentrations, pellets were suspended by vigorous mixing in respectivebuffer. Final cell, particle, buffer and salt concentrations wereadjusted by adding 10× stock solutions to 50% suspensions and dilutionwith ddH2O to working volume (20 ml). Incubation was performed for up to3 hours at room temperature (˜23° C.) in stirred beakers (mixing) or intubes (static). Both had similar height to diameter ratios. Mixingintensity was adjusted to 800 rpm with a convenient magnetic stirrer atbottom. For quantification of released protein aliquots of 1 ml werediluted 1:2 with respective buffer. Samples were centrifuged thereuponat 8000 rcf for up to 10 min (˜23° C.) and supernatants collected forfurther investigations.

Example 2.2 Extraction of Target Proteins by HPH

E. coli (HMS174) cells were cultivated in fed batch at 37° C.(GFPmut3.1) and 30° C. (SOD). Cell were suspended in buffer (50 mM Tris,pH 8.0, 100 mM NaCl) to 25% v/v and disrupted by high pressurehomogenisation (Niro-Soave Panda 2k, 2x passes at 100 MPa).

Example 3: Quantification of Target Proteins by Fluorescence Example 3.1Fluorescence

GFPmut3.1 standard (>95%) was prepared by sequential purification fromclarified E. coli homogenate (centrifuged for 60 min at 10000 rcf and0.2 micro filtered) with anion exchange (AIEX CaptoQ), hydrophobicinteraction (ButylSepharose) and gel filtration (SuperdexG75 prep.grade) chromatography. Concentration was determined from absorbance at280 nm (denatured in 8M Urea, 10 min at 100° C.). Equivalentfluorescence was determined from standard calibration at 485 nm(excitation) and 535/20 nm (emission) on plate reader (Tecan GENiosPro).

Recovery efficiency (% Recovery) of GFP was quantified relative to HPHcell disruption (2 passages at 100 MPa as described in Example 2.2).

Example 3.1.1 Cell Concentration

The recovery of GFP from E. coli cells recovered by adsorption on groundMARATHON® A2 (MA2) and elution with NaCl was determined. Cells wereseparated from cell broth by centrifugation and suspended in 50 mM TRISat pH 8.0 at volumetric concentrations of 20% (v/v), 6% (v/v), and 1%(v/v). Cell suspension was mixed with various volumetric ratios of resin200%, 100%, 50% and 0% and incubated statically for 1 h. Elution wasperformed by 1:2 dilutions of suspensions aliquots with 2M NaCl. GFP inaqueous phase was quantified by fluorescence. Ground MARATHON® MSC(MMSC) was used at same conditions for comparison with cationic resin.

FIG. 2 shows that at 6% (v/v) cell concentration a GFP recovery of 100%was achieved. At 20% (v/v) cell concentration the amount of extractedprotein decreased below 50%. In contrast, adding lower amounts ofmicroparticles to 20% (v/v) cell suspension resulted in higher recovery,up to 45%. At 1% amount of recovered GFP decreased proportionally withamount of microparticles. With no microparticles added, amount of GFP insupernatants was below 20%. No effect of protein extraction and celldisruption could be observed by incubation of cells with groundMARATHON® MSC (cation exchanger) at same conditions.

Example 3.1.2 Volumetric Ratio of Resin:Cells

The recovery of extracted GFP from E. coli cells recovered by adsorptionon ground MARATHON® A2 (MA2) and elution with NaCl at differentvolumetric ratio of resin:cells was determined. Cells were separatedfrom cell broth by centrifugation and suspended in 50 mM TRIS at pH8.0at 10% (v/v). The cell suspension was mixed with various volumetricratios of resin 200%, 100%, 70%, 50%, 30% and 0% and incubatedstatically for 1 h. Elution was performed by 1:2 dilutions ofsuspensions aliquots with 2M NaCl. GFP in aqueous phase was quantifiedby fluorescence.

FIG. 3 shows that after one hour of incubation, almost 100% of GFP wasrecovered by desorption from ground MARATHON® A2. Less than 20% of totalsoluble GFP was extracted from cells without MARATHON® A2.

Example 3.1.3 pH and Incubation Time

The recovery of extracted GFP from E. coli cells recovered by adsorptionon ground MARATHON® A2 (MA2) and elution with NaCl at different pHvalues and different incubation times was determined. The pH of cellbroth suspensions was adjusted to 7.5, 8.0 and 8.5 by adding NaOH. Cellsuspension was mixed with a volumetric ratio of 100% of resin andincubated statically for 3 h. Periodically (10, 60, 120, 180 min)elution was performed by 1:2 dilutions of suspensions aliquots with 2MNaCl. GFP in aqueous phase was quantified by fluorescence. After amaximum incubation time of 3 h approx. 40% and 70% of total soluble GFPcould be recovered at pH 7.5 and 8.0 from cell broth. 100% recovery wasachieved only at pH8.5.

Example 3.1.4 pH, Volumetric Ratio Resin:Cells and Incubation Time

The influence of the volumetric resin:cell ratio between 0-100% wasfurther investigated. The pH of cell broth suspensions was adjusted to7.5, 8.0 and 8.5 by adding NaOH. Cell suspension was mixed with variousvolumetric resin:cell ratios of 100%, 70%, 50%, 30% and 0% and incubatedstatically for 3 h. Periodically (10, 60, 120, 180 min) elution wasperformed by 1:2 dilutions of suspensions aliquots with 2M NaCl. GFP inaqueous phase was quantified by fluorescence.

FIGS. 4A-4C show that while the pH had a significant influence on themaximum recovery, the effect of added resin amount was negligible. 70%resin:cells ratio (v/v) at pH 8.5 after 2 hours of static incubation wasconsidered to be efficient for disruption of 10% (v/v) of cellsuspension and recovery of GFP directly from cell broth.

Example 3.1.5 Salt Concentration and GFP Adsorption Capacity

The recovery of extracted GFP from E. coli cells recovered by adsorptionon ground MARATHON® A2 (MA2) and elution with NaCl at different NaClconcentrations (0-500 mM) was determined. Cells were separated from cellbroth by centrifugation and suspended in 50 mM TRIS at pH8.0 at 10%,v/v. NaCl concentration of suspensions was adjusted with 2M NaCl buffersolution. Cell suspension was mixed with various volumetric ratios of100%, 70%, 50%, 30% and 0% of resin:cells and incubated statically for 1h. Elution was performed by 1:2 dilutions of suspensions aliquots with2M NaCl. GFP in aqueous phase was quantitated by fluorescence.

FIG. 5 shows that up to 100% GFP recovery was achieved at 0 mM NaCl.

The amount of GFP bound to microparticles was investigated by comparisonof concentration in aqueous phase (supernatant) before and afterdesorption (FIG. 6). The difference of GFP quantity was considered to beadsorbed to resin. Trends were fitted with the standard Langmuirequation.

Example 3.1.6 Extraction with Anionic Chelating Resin

Protein extraction was also demonstrated from incubation of E. colicells with chelating microparticles (FIGS. 7A-7B). In contrast tostrongly basic quaternary groups of MARATHON® A2, iminodiacetic acidgroups of self-made microparticles from AMBERLITE® IRA 748 are cationexchangers with high affinity to multivalent cations. In that case noflocculation of suspension had been observed.

GFP extraction from cell suspension (E. coli) with chelating (groundAMBERLITE® IRA 748) and cationic microparticles (ground MARATHON® A2)was determined after incubation in 50 mM TRIS buffer at pH8.0 at 30% and70% v/v volume ratios of resin:cells (5% v/v) and subsequent elutionwith 1M NaCl and compared to GFP extraction under the same conditionswithout elution with salt (FIGS. 7A-7B). Further, GFP extraction understirring and static incubation conditions was determined (FIGS. 7A-7B).GFP was not adsorbed on chelating microparticles in contrast to groundMA2 wherein protein had been recovered by desorption. The protein yieldfrom seems not to be influenced by static or stirring conditions duringincubation with chelating microparticles at performed scale.

Example 3.1.7 Influence of Elution Conditions on Protein Extraction

GFP extraction from cell suspension (E. coli) with chelating (groundAMBERLITE® IRA 748) and cationic microparticles (ground MARATHON® A2)was determined after static incubation for 2 hours in 50 mM TRIS bufferat pH8.0 at 30%, 70% and 100% v/v volume ratios of resin:cells (5% v/v)and subsequent elution with 1M NaCl and compared to GFP extraction underthe same conditions without elution with salt. GFP extraction yields upto 100% were obtained by incubation of E. coli cells for 2 hours at pH8.0, 50 mM TRIS with ground AMBERLITE® IRA 748 (FIG. 8). The yield ofrecovered GFP increased with volumetric ratio of chelatingmicroparticles added.

Example 3.1.8 Influence of Cell Concentration on Protein Extraction

Protein (GFP) extraction from cell suspension (E. coli) with chelatingresin (ground AMBERLITE® IRA 748) was determined after incubation in 50mM TRIS buffer at pH 8.0 in stirred beakers with 70% v/v volume rationof resin:cells at 20%, 15%, 10% and 5% volumetric cell concentration andno elution with salt (FIG. 9). Higher cell concentrations in suspensionfavourably influenced the extraction yield for chelating miroparticles.A GFP extraction kinetic of extracted protein amount in relation to drybiomass (d.m.) of E. coli was determined (FIG. 10).

Example 4: Quantification of Target Proteins by SDS Page

Heavy solid fraction of E. coli homogenate was separated bycentrifugation at 4000 rcf for 15 min. Supernatant was transferred andlight fraction was collected after 60 min centrifugation at 4000 rcf.Pellet was suspended in deionized water and washed sequentially twotimes as before. Reference samples of cell debris and crude homogenatewere diluted 1:5 with 10M Urea, pH 8.0 and incubated for one hour on arotatory shaker.

All samples were heated for 10 minutes at 100° C. in 1:4 SDS Samplebuffer and 0.2M DTT. Electrophoresis was performed on 8-12%polyacrylamide gels in MES-SDS running buffer at 200V and a maximum of400 mA for 60 minutes. Staining was performed with CoomassieR250 andBismarkBraunR (Choi et al. 1996). Densitometry was performed by opticalgel scanning (Epson Perfection Scan V770, 600 dpi, 16 bit grayscale) andevaluation with LumiAnalyst (v3.0 Roche Diagnostics) software.

Example 4.1 SOD Extraction

Protein extraction was demonstrated from incubation of E. coli cellswith cationic microparticles obtained from AMBERLITE® IRA458. Incontrast to styrene divinylbenzene (PS/DVB) matrix of MARATHON® A2,microparticles obtained from acrylic resin contained more water at samepacked bed volume. Flocculation was apparently stronger with acrylicmicroparticles (bigger flocks and faster sedimentation) but this was notinvestigated in detail. Additionally target protein was superoxidedismutase (SOD) instead of GFP.

Example 4.1.1 Protein Yield at Varying Incubation Conditions

Protein (SOD) extraction yield was determined from cell suspension (E.coli) with acrylic resin (ground AMBERLITE® IRA458) after staticincubation in 50 mM TRIS buffer at pH8.0 in tubes at 50, 70 and 100% v/vvolume ratio of resin:cells (10% v/v) and subsequent elution with NaCl.Protein amount was estimated by densitometry from standard calibrationon SDS-Page (FIGS. 11A-11D). The kinetic of SOD release was almostfinished after 1 hour of static incubation with acrylic resin. SODadsorbed to acrylic microparticles was recovered with 0.5 M and 1.0 MNaCl to an equal amount.

Example 4.1.2 SOD Recovery from E. coli Homogenate

Recovery of SOD from E. coli homogenate obtained from 10% (v/v) cellsuspension was determined after incubation with AMBERLITE® IRA 458microparticles (100%, 70%, 50 v/v volumetric resin:cells ratio) aftershort mixing and incubation (50 mM TRIS, pH 8.0) and subsequent elutionwith NaCl (0.0 M, 0.5 M, 0.1 M). SOD adsorbed to AMBERLITE® IRA 458microparticles was recovered in equal amounts at elution in 0.5 M and1.0 M (FIG. 12).

Example 4.1.3 SOD Adsorption Capacity

The amount of SOD bound to microparticles was investigated.

SOD standard (>95% appreciated by Coomassie staining of SDS-Page) wasprepared by extraction from E. coli cells with A2 microparticles anddesorption after 2 h of incubation at 70% v/v (resin/cells) in 50 mMTRIS buffer at pH 8.0 with 0.2M NaCl and subsequently purified by gelfiltration (SuperdexG75 prep. grade from GE). Concentration wasdetermined from absorbance at 280 nm (denatured in 8M Urea, 10 min at100° C.). Concentration of samples was evaluated by densitometry from 5point calibration on SDS-page. Concentrations of SOD samples obtained byextraction and homogenization were compared by integration relatively tostandard.

Example 5: Non-Target Protein Extraction

Purity of extracted GFP by adsorption on ground MARATHON® A2 was checkedby SDS-Page in comparison with homogenate. After HPH the heavy solidfraction (containing e.g. GFP inclusion bodies) of E. coli homogenatewas separated by centrifugation at 4000 rcf for 15 min. Supernatant wastransferred and light homogenate fraction was collected after 60 mincentrifugation at 4000 rcf. The light homogenate fraction contains celldebris and membrane proteins such as outer membrane protein A (OmpA) andacts as an indicator of cell disruption. Pellet was suspended indeionized water and washed sequentially two times as before. Referencesamples of cell debris and crude homogenate were diluted 1:5 with 10Murea, pH 8.0 and incubated for one hour on a rotatory shaker. SDS-Pagewas performed with crude homogenate, homogenate supernatant and elutedprotein extracted by incubation of cells with 50% v/v ground MARATHON®A2 at pH8.0 for 3 h in 50 mM TRIS. Aliquots of suspension were eluted by1:2 dilutions in 200, 250, 300, 350, 400, 450, 500 and 1000 mM NaCl.SDS-Page of all samples was performed at same dilution ratio anddensitometry of Coomassie staining was used for appreciation of proteinamounts. Washed cell debris was applied as reference for major membraneprotein at ˜39 kDa (OMP).

Within desorption conditions at 300 mM NaCl, only 15% respectively 17%of non-target proteins in comparison with crude and cleared homogenatewere detected after extraction. At same conditions 6% and 12% of proteinfrom light homogenate fraction were respectively found in supernatant

Further, homogenate supernatant and eluted protein were compared afterincubation of cells with 70% v/v ground AMBERLITE® IRC 748 and groundMARATHON® A2 at pH 8.0 in 50 mM TRIS for 2 hours at mixing at staticconditions, respectively. Aliquots of suspension were diluted 1:2 in 50mM TRIS buffer or 1 M NaCl. SD S-Page of all samples was performed atthe same dilution ration and densitometry of Coomassie staining was usedfor appreciation of protein amounts.

GFP recovery was evaluated by densitometry of SDS-Page (Coomassie) andcompared to eluted host cell proteins, in particular membrane proteins(FIGS. 13, 14, 15). Extracted proteins were compared after incubation ofcells with 50% v/v ground MARATHON® A2 at pH8.0 for 3 h in 50 mM TRIS.Aliquots of suspension were eluted by 1:2 dilutions in 200, 250, 300,350, 400, 450, 500 and 1000 mM NaCl. SDS-Page of all samples wasperformed at same dilution ratio and densitometry of Coomassie stainingwas used for appreciation of protein amounts (FIG. 13). GFP wasrecovered to almost 100% in supernatant at 300 mM NaCl from MARATHON®A2. Desorption at higher salt concentrations caused recovery of higheramounts of non-target and light fraction proteins (FIG. 14). On average15% light fraction protein and 20% of non-target proteins were extractedrespectively from cells with microparticles in comparison with highpressure homogenization in supernatant.

The profile of detected proteins by Coomassie staining showed puritydifferences between the supernatant of cells disrupted by HPH and byextraction with microparticles. The protein profile of homogenatesupernatant and eluted protein with 300 mM NaCl after incubation ofcells with 50% v/v ground MARATHON® A2 at pH8.0 in 50 mM TRIS for 3 hisshown in FIG. 15. SDS-Page of samples was performed at same dilutionratio and densitometry of Coomassie staining was used for appreciationof relative protein amounts. Relative purity for GFP was 9.8% inhomogenate and 37.4% in elute at 300 mM NaCl and on average at33.2%±2.9% after extraction.

In another approach, the protein profile of homogenate supernatant andextracted protein was determined after incubation of cells with 70%ground AMBERLITE® 748 at pH 8.0 in 50 mM TRIS for 2 hours. SDS-Page ofsamples was performed at the same dilution ration and densitometry ofCoomassie staining was used for appreciation of relative proteinamounts. Relative purity of GFP was 12.4% in homogenate and 38.4% insupernatant and on average at 41.6%±2.7% after extraction.

The non-target protein reduction during extraction kinetic of protein(SOD) from cell suspension (E. coli) with cationic resin (MARATHON® A2)after static incubation in 50 mM TRIS buffer at pH 8.0 (10% v/v) andsubsequent elution with NaCl was determined. Non-target proteins amountwas estimated by densitometry from SDS-Page. The purity of SOD extractedfrom cells was two times higher than of that captured from homogenate bystyrenic micro particles.

Example 6: SOD Activity

Enzymatic activity of SOD extracted from cells by styrenic cationicmicroparticles MARATHON® A2 (MA2) was determined after extraction fromcell suspension (E. coli) and homogenate after static incubation in 50mM TRIS buffer at pH 8.0 at 70% v/v volume ration of resin:cells (10%v/v) and subsequent elution with 0.5 M NaCl. Supernatants werecentrifuged (1 ml, 30 min, 23° C. and 16000 rcf), filtered (PVDFmembrane, 0.2 μm) and diluted in 1:10 steps with respective buffer to aneffective measurement range. SOD activity was determined by 19160 SODdetermination kit purchased from Sigma. Enzymatic activity of SODextracted from cells by MA2 microparticles was on average increased byone unit compared to that captured from homogenate.

Example 7: Contaminants

DNA was quantified with Quant-iT™ PicoGreen® dsDNA Assay Kit purchasedfrom Invitrogen. Endotoxin was quantified with PyroGene™ RecombinantFactor C Assay purchased from Lonza. All supernatant samples werecentrifuged (1 ml, 30 min, 23° C. and 16000 rcf), filtered (PVDFmembrane, 0.2 μm) and diluted in 1:10 steps with respective buffer to aneffective measurement range Measurements were performed accordingly torespective kit instruction notes on plate reader (GENios Pro or Infinite200M from Tecan).

Example 7.1 dsDNA

The reduction of dsDNA was determined during the extraction of protein(SOD) from a cell suspension (E. coli) with acrylic (AMBERLITE® IRA458)and cationic resin (MARATHON® A2) after static incubation in 50 mM TRISbuffer at pH8.0 at 50, 70, 100% v/v volume ratio of resin to cells (10%v/v) and subsequent elution with NaCl. DNA was quantified as describedabove. Up to 10² less dsDNA was released by incubation of cells withmicroparticles. At protein elution conditions (0.5 M NaCl), dsDNAreduction in the supernatant was between 10² and 10³ (FIGS. 16A-16F).

Example 7.2 Endotoxin

Endotoxin reduction was determined during extraction kinetic of protein(SOD) from cell suspension (E. coli) with cationic resin (MARATHON® A2).after static incubation in 50 mM TRIS buffer at pH 8.0 (10% v/v) andsubsequent elution with NaCl. Endotoxin amount was quantified asdescribed above.

At extraction conditions 10³ less endotoxin was released to thesupernatant in comparison to adsorption from the homogenate (FIG. 17).At 0.5 M NaCl and 1.0 M NaCl endotoxin was about 10× reduced.

Example 8: Microscopy Example 8.1 Atomic Force Microscopy (AFM)

Positively charged microscopic slides were sequentially treated withsucrose (50 mM) solution, deionised water and diluted suspension ofmicroparticles. After each step drying was performed at 65° C. for 24 h.Measurements were carried out at the Department of Nanobiotechnology,BOKU (Dr. Gerhard Sekhot).

The open source software Gwyddion (v2.60) was used for visualisation ofAFM data. An edged surface topography was apparently generated as aconsequence of mechanical abrasion of original beads. Ground particleshad similar surface topography (FIG. 18).

Images generated by AFM (FIG. 19) of E. coli before and after incubationwith ground MARATHON® A2 indicated that protein was extracted withoutfragmentation of cells.

Example 8.2 Optical Microscopy

Confocal and fluorescence microscopy were performed at Vienna Instituteof Biotechnology (VIBT) on Leica “Live Cell” wide-field microscope.Samples were diluted to ˜1% v/v solids concentration in respectivebuffer and visualized at 1000× magnification (Leica HCX PL APO 100×1.4oil).

Bright field (BF), differential interference contrasts (DIC) andfluorescence microscopy was performed at 1000× magnification. Cells weremobile and bright fluorescent at default excitation and emissionwavelengths of GFP. Cells were immobilized by adsorption onmicroparticles (FIG. 20).

Example 9: Cell Viability Example 9.1 BacLite

Viability of E. coli cells (10% v/v) after 2 hours of static incubation(50 mM TRIS, pH 8.0) with MARATHON® A2 microparticles (70% volumetricresin:cells ratio) and 1:10 dilution in physiological buffer. Cells werestained with “BacLite” (Invitrogen). Live (green) and dead (red) cellsare shown in FIG. 21.

Example 9.2 Koch's Plates

Cell viability was determined after 3 h of static incubation of E. colicells (10% v/v) with microparticles obtained from ground MARATHON® A2and AMBERLITE® IRA 458 (70% v/v resin:cells ratio) in 50 mM TRIS and pH8.0. Cells (E. coli GFP) and cells/resin suspensions (1 ml aliquots)were diluted progressively and mixed vigorously 1:10 in sterile 0.9% w/wNaCl. Nutrient agar (NA) for microbiology Merck 20 g/l in ddH₂O washeated in a steam autoclave at 121° C. for 15 min. Aliquots (1 ml) ofdiluted suspensions were poured into culture dishes and mixed with 55°C. tempered NA solution. Gel formation was considered completed after 30min at room temperature and plates were incubated overhead at 37° C. for30 h.

Up to 10³ more colony forming units (CFU) were determined afterincubation with cationic microparticles. Relevant CFU values formicroparticles were obtained after 1000 dilution in physiological bufferwhich indicated that adsorbed cells could not propagate.

Identity of cells was determined by optical microscopy from 10×dilutions of cells without resin and cells at 1000× dilutions after GFPextraction with cationic microparticles. Size, shape and fluorescencecorrespond to E. coli GFP.

Example 10: Hydrophobic Microparticles

Preparation of Hydrophobic Microparticles

Adsorbent type resin provided by DOW: AMBERLITE® XAD4, AMBERLITE® XAD7HPand AMBERLITE® XAD761 were purchased from Sigma Aldrich, Vienna,Austria, 2011.

Resins were grinded overnight (20 g for ˜12 h) with an electric motordriven, ceramic coated mortar. Grinded resins were suspended in water(˜10% v/v and ad. 50 ml). Supernatant was centrifuged for 30 min at4000×g (equivalent with relative centrifugal force). Resins werere-suspended in 2 M sodium chloride (50 ml) centrifuged for 1 min(4000×g). Pellet of 1 min centrifugation was discarded. Supernatant weretransferred and centrifuged again for 30 min (4000×g). Supernatant wasdiscarded. Grinded resins were re-suspended (1:2) in water andtransferred to tubes. Resins were centrifuged at 4000×g, supernatant wasdiscarded and resin was re-suspended in 50 ml of aqueous washingsolution.

Wash sequence was:

1×50% EtOH (dilution of organic residues)

3× deionized water (dilution of EtOH)

Particle Size of Hydrophobic Microparticles

Particle size distributions of microparticles were calculated fromequivalent circular diameter by measurement of bright field microscopyprojections of approximately 500 particles at 1% v/v and 600× foldmagnification.

General Protocol for Microparticles and Conventional ChromatographicMedia Adsorption/Desorption

Adsorption and desorption studies were performed in 1 mL batches(homogenate or standard protein solution). Various amounts of 50% (v/v)microparticles suspensions (in μL), were added to protein solutions in 2mL tubes. Dilution factor concerning protein concentration andconductivity was taken into account.

Microparticles suspensions were incubated for 30 minutes, conventionalchromatographic media suspension for 12 hours. Afterwards microparticlesor conventional chromatographic media were centrifuged at 7000×g for 10min and elution of bound protein was performed by addition of 1 mLelution buffer, vigorous mixing and incubation for 30 min. In some casesa second washing step with elution buffer was included. After elutionmicroparticles or conventional chromatographic media were centrifugedagain as before. Concentration of protein in supernatants was quantifiedby photometric analysis and purity of target protein was checked bySDS-PAGE.

Example 11: Recovery of Acidic Biomolecules

Batch adsorption of recombinant GFP from E. coli crude homogenate wasperformed using Microparticles (MPs) (ground chromatography resinMARATHON® A2 (MA2)) with the following steps. This Example uses GFP asacidic intracellular soluble protein.

E. coli strain HMS174(DE3)(pET11aGFPmut3.1) was fermented in a 5 L scalefed-batch process. The expression of the intracellular soluble targetprotein GFP (green fluorescent protein) was induced using IPTG(Isopropyl β-D-1-thiogalactopyranoside).

Harvest and Homogenization: The E. coli suspension (biomass content ˜30%wt) was cooled to 4° C. and centrifuged at 15000 g for 20 min. Thesupernatant was discarded and the cell pellet was further processed. Thecell pellet was resuspended in 50 mM Tris, pH 7.5 and diluted to abiomass content 20% wt cells/buffer. Cells were disrupted by highpressure homogenization at 1000 bar for two passages producing the crudecell lysate.

For the batch adsorption from E. coli lysate it is also possible to usefrozen biomass. In this case the biomass (20% w/v) is resuspended in 50mM Tris, pH 7.5, and same disruption procedures can also be applied asis the case for fresh fermented E. coli cells.

Capture of the Target Protein

The batch adsorption was performed in tubes in small scale of 1 mLvolume as well as in scales up to 100 mL in glass beakers at roomtemperature (rt). To the crude cell lysate MA2 was added (1 μL of 50%v/v % MA2 were added per 1 μg cell pellet) and mixed for ˜5 s in a labvortex or in bigger scale with an overhead stirrer for ˜30 s. Duringmixing the flocculation took place and the MA2 bound to the targetprotein as well as impurities like DNA, hcps (host cell proteins) andcell fragments. After the flocculation the samples were centrifuged for3 min at 13400 g or filtrated using a 0.2 μm filter plate at 1.5 bar ina dead-end filtration with overhead pressure. The supernatant wasdiscarded and the pellet/filter cake further processed for the washstep.

Flocculate Wash

The pellet of the flocculate was resuspended in a 50 mM Tris wash bufferwith 75 mM NaCl at pH 7.5. After short incubation the flocculate wasseparated using centrifugation (13400 g for 3 min). When the separationtook place using a filtration process the filter cake was notresuspended but washed by filtrating the wash buffer through the filtercake (0.2 μm filter plate at 1.5 bar). The supernatant was discarded andthe pellet/filter cake further processed for the elution step. The lowsalt concentration is able to elute impurities with low bindingstrength.

Elution: For the elution step the washed flocculate was resuspended in50 mM Tris buffer containing 400 mM NaCl at pH 7.5. The flocculate wasmixed in a tumbler for 5 min. At 400 mM NaCl concentration the targetprotein elutes from the MPs and is now in the supernatant. Thesupernatant was separated from the flocculate using centrifugation(13400 g for 3 min) or per dead-end filtration (0.2 μm filter plate at1.5 bar). The pellet/filter cake containing the MPs with boundimpurities was discarded and the supernatant containing the protein ofinterest (GFP) was processed further.

Elution profile of the GFP is shown in FIG. 22. The lysate sample can beseen on lane 1 which is the starting material for the purificationprocess. Lane 2 is the marker (Mark12™). Lane 3 shows the supernatantafter capture. The missing GFP band shows that all GFP molecules arebound to the MPs. On lane 4 is the wash step. The elution takes placebetween 100-400 mM NaCl. Pooling of the elution samples provided a yieldof 98% and lane purity about 60% for GFP.

Example 12: Recovery of Basic Biomolecules Using Positively ChargedMicroparticles

This example demonstrates the recovery of recombinant expressed basicproteins using MPs from ground MARATHON® A2 (MA2) resin from cellhomogenate. The protein Interferon Gamma, IFN-γ, functions as an examplefor an intracellular soluble expressed basic protein. This example showsthat the positively charged exchange resin can be used for biomoleculerecovery by binding to unwanted cellular structures and intracellularmaterial (referred to as negative purification).

IFN-γ was expressed intracellularly soluble in E. coli by fed-batchfermentation.

Harvest and Homogenization

Frozen biomass (20% w/v) of cells which express Interferon Gamma IFN-γwas used and resuspended in lysis buffer (20 mM Tris, 10 mM EDTA, 1 MUrea, 0.1% beta-mercaptoethanol). The cells were disrupted by highpressure homogenization at 950 bar for three passages producing thecrude cell lysate. The cell disruption would also work with freshbiomass.

Negative Purification of the Target Protein

The batch adsorption was performed in tubes in small scale of 2 mLvolume at room temperature (rt). The crude cell lysate was mixed withMA2 (50% v/v) and mixed for ˜5 s in a lab vortex (0.7 μL of 50% v/v MA2added per 1 μg wet cell pellet). During mixing the flocculation takesplace where MA2 binds negatively charged impurities like DNA, hcps (hostcell proteins) and cell fragments. After the flocculation the sampleswere centrifuged for 3 min at 13400 g or filtrated using a 0.2 μm filterplate at 1.5 bar in a dead-end filtration with overhead pressure. Thepellet/filter cake containing the MPs with bound impurities wasdiscarded and the supernatant containing the protein of interest (IFN-γ)was processed further.

Elution profile of the Interferon Gamma IFN-γ is shown in FIG. 23. Onlane 1 is the marker Mark12′ followed by BSA on lanes 2-4 forquantification purposes. On lane 5 is the cell homogenate. On lane 6 isthe supernatant sample the addition of positively charged microparticles(MARATHON® A2) that bind HCPs DNA and cell fragments. Lane 7 shows anexemplary pellet wash & strip with 1000 mM NaCl to elute the impurities.The yield is up to 99% with a purity of 30%.

Example 13: Recovery of Acidic Biomolecules Using Positively ChargedMicroparticles from E. coli Cells

This example shows the extraction of an acidic intracellular solubleprotein from intact E. coli cells using positively charged MPs preparedfrom MA2. In this example the used target protein is GFP. The proteinextraction was shown using two different E. coli strains, HMS174(DE3)and BL21 each with a different GFP molecule GFPmut3.1 and GFP.1respectively. Two variants for the protein extraction were carried out

Example 13.1

E. coli strains HMS174(DE3)(pET11aGFPmut3.1) and BL21(pB11KT7ix.1_GFP.1)were fermented in a 5 L scale fed-batch process. The expression of theintracellular soluble target protein GFP (green fluorescent protein) wasinduced using IPTG (Isopropyl β-D-1-thiogalactopyranoside).

Cell Harvest & Wash

The cells were harvested and stored overnight (˜12 h) at 4° C. inFLEXBOY® Bags. The E. coli suspension (biomass content ˜30% wt) wascooled to 4° C. and centrifuged at 15000 g for 20 min and laterresuspended with a 50 mM Tris buffer at pH 7.5 while containing the samebiomass content.

Cell Flocculation and Protein Recovery

Example 13.1 uses reduced amount of MPs (85 μL MA2 (50% v/v) per 1 mLcell suspension at 30% wet biomass content) to bind and flocculate theE. coli cells. While the MPs were in contact with the cells, theextraction took place and the target protein (here GFP) were be releasedand accumulated in the supernatant. After an incubation of 2-3 h theextraction was complete and the flocculated cells were separated usingdead-end filtration (0.2 μm filter plate at 1.5 bar) or centrifugation(13000 g for 3 min). The cell pellet/filter cake was discarded and thesupernatant containing the target protein was further processed.

Example 13.2

In this Example more microparticles were added to the cell suspension.The higher amount of microparticles allowed the binding and extractionof the target protein directly.

E. coli strains HMS174(DE3)(pET11aGFPmut3.1) and BL21 (pB11KT7ix.1_GFP.1) were fermented in a 5 L scale fed-batch process. The expressionof the intracellular soluble target protein GFP (green fluorescentprotein) was induced using IPTG (Isopropyl β-D-1-thiogalactopyranoside).

Cell Harvest & Wash

The cells were harvested and stored overnight (˜12 h) at 4° C. inFLEXBOY® Bags. The E. coli suspension (biomass content ˜30% wet) wascooled to 4° C. and centrifuged at 15000 g for 20 min and laterresuspended with a 50 mM Tris buffer at pH 7.5 while containing the samebiomass content.

Cell Flocculation and Protein Recovery

Example 13.2 uses a higher amount of microparticles (350 μL MA2 (50%v/v) per 1 mL cell suspension at 30% wet biomass content) compared toExample 13.1 to bind and flocculate the E. coli cells. While themicroparticles were in contact with the cells, the extraction of thetarget protein took place and the proteins were directly bound by theMPs. After incubation between 1.5 to 2.5 h the extraction was completed.The supernatant was separated using dead-end filtration (0.2 μm filterplate at 1.5 bar) or centrifugation (13000 g for 3 min). The supernatantwas discarded and the pellet of the flocculate the target protein wasfurther processed.

Flocculate Wash

The pellet of the flocculate was resuspended in a 50 mM Tris wash bufferwith 75 mM NaCl at pH 7.5. After short incubation the flocculate wasseparated using centrifugation (13400 g for 3 min). When the separationtook place using a filtration process, the filter cake was notresuspended but washed by filtrating the wash buffer through the filtercake (0.2 μm filter plate at 1.5 bar). The supernatant was discarded andthe pellet/filter cake further processed for the elution step. The lowsalt concentration is able to elute impurities with low bindingstrength.

Elution

For the elution step the washed flocculate is resuspended in 50 mM Trisbuffer containing 400 mM NaCl at pH 7.5. The flocculate was mixed in atumbler for 5 min. At 400 mM NaCl concentration the target proteinelutes from the MPs and is now in the supernatant. The supernatant wasseparated from the flocculate using centrifugation (13400 g for 3 min)or per dead-end filtration (0.2 μm filter plate at 1.5 bar). Thepellet/filter cake containing the MPs with bound impurities wasdiscarded and the supernatant containing the protein of interest (GFP)was processed further.

FIG. 24 is a bar graph showing the GFP yield from Example 13.2 for twodifferent incubation times: 1 h and 2 h. 1 shows the GFP amount in thecapture supernatant. 2 shows the GFP amount in the wash buffer in thewashing step of the flocculate. No GFP leakage can be seen in 1 or 2. 3shows the elution with 400 mM NaCl in 50 mM Tris, where the GFP waseluted from the MPs and accumulated in the supernatant. 4 shows themaximum amount of GFP in the cells which were lysed using a chemicaldisruption method.

Example 14: Recovery of a Basic Protein from Cells Using PositivelyCharged Microparticles

Positively charged microparticles are prepared from grinding DOWEX® M-A2anion exchange resins to obtain microparticles as described in Example1.

IFN-γ was expressed inracellularly soluble in E. coli by fed-batchfermentation.

Cell Harvest & Wash

The cells were harvested and stored overnight (˜12 h) at 4° C. inFLEXBOY® Bags. The E. coli suspension (biomass content ˜30% wt) wascooled to 4° C. and centrifuged at 15000 g for 20 min and laterresuspended with a buffer (20 mM Tris, 10 mM EDTA, 1 M Urea, 0.1%beta-mercaptoethanol) while containing the same biomass content.

Cell Flocculation and Protein Recovery

MPs (85 μL MA2 (50% v/v) per 1 mL cell suspension (at 30% wet biomasscontent) are added to the cell suspension to bind and flocculate the E.coli cells. While the MPs were in contact with the cells, the extractiontook place and the target protein (here IFN-γ) was released andaccumulated in the supernatant. After an incubation of 2-3 h theextraction was complete and the flocculated cells were separated usingdead-end filtration (0.2 μm filter plate at 1.5 bar) or centrifugation(13000 g for 3 min). The cell pellet/filter cake was discarded and thesupernatant containing the target protein was further processed.

Example 15: Recovery of Basic Proteins from Cells Using NegativelyCharged, Chelating Microparticles

Microparticles are prepared from chelating cation exchange resinAMBERLITE® IRC748.

IFN-γ was expressed inracellularly soluble in E. coli by fed-batchfermentation.

Cell Harvest & Wash

The cells were harvested and stored overnight (˜12 h) at 4° C. inFLEXBOY® Bags. The E. coli suspension (biomass content ˜30% wt) wascooled to 4° C. and centrifuged at 15000 g for 20 min and laterresuspended with a buffer (20 mM Tris, 10 mM EDTA, 1 M Urea, 0.1%beta-mercaptoethanol) while containing the same biomass content.

Cell Flocculation and Protein Recovery

MPs (85 μL AMBERLITE® IRC748 (50% v/v) per 1 mL cell suspension (at 30%wet biomass content) are added to the cell suspension to bind andflocculate the E. coli cells. While the MPs were in contact with thecells, the extraction took place and the target protein (here IFN-γ) wasreleased and accumulated in the supernatant. After an incubation of 2-3h the extraction was complete and the flocculated cells were separatedusing dead-end filtration (0.2 μm filter plate at 1.5 bar) orcentrifugation (13000 g for 3 min). The cell pellet/filter cake wasdiscarded and the supernatant containing the target protein was furtherprocessed.

Example 16: Preparation of Microparticles from Ion Exchange Resins

Different types of ion exchange resins were purchased from Sigma Aldrichand DIAION®.

The following anion exchanger resins were used: AMBERLITE® IRA-400,AMBERLITE® IRA-743, DOWEX® 1×2-100, DOWEX® 1×2-400, DOWEX® 1×8-100,MARATHON® A2, DIAION® SA20A, DIAION® SA10A, DIAION® SA312.

The following cation exchanger resins were used: DOWEX® 50 WX2-100,DOWEX® 50 WX8-100, MARATHON® C, MARATHON® MSC, DIAION® PK216, DIAION®SK110.

Resins were wet ground (20 g for ½ h) in a coated ceramic mortar by handfor ca. 30 min. Ground resins were suspended in water (ad 50 ml). Aftera period (ca. 96 h) supernatant of resins sediment was transferred totubes. Supernatant was piecewise (1 ml) centrifuged for 15 min at 7000rcf (relative centrifugal force) until ca. 200 μl resin was collectedper tube. Resins were re-suspended in 2M Sodium chloride (1.5 ml) ancentrifuged for 1 min (7000 rcf). Pellet of 1 min centrifugation wasdiscarded (excepting AIVIBERLITE® IRA-743). Supernatant were transferredand centrifuged again for 15 min (7000 rcf). Supernatant of 15 mincentrifugation was discarded. Micro particles (ca. 200 μl) were alsocentrifuged in 2M Sodium chloride. Micro particles (ca. 150 μl) andother ground resins (50-200 μl) were re-suspended (1:4) in water andtransferred in portions (50 μl resin) to tubes.

Aliquots of resin were centrifuged at 7000 rcf, supernatant wasdiscarded and resin was re-suspended in 20 fold volume (about 1 ml) ofaqueous washing solution. Time of incubation in solution was 30 min.

Wash Sequence:

-   -   1×50% EtOH (dilution of organic residues)    -   3× deionized water (dilution of EtOH)    -   Check for near neutral pH    -   re-suspension of micro particles and ground resin in deionized        water (about 70% v/v)    -   Resins were equilibrated in corresponding buffer used for        specific experiments.

Determination of Particle Size Using Optical Microscopy

Particle size of the prepared microparticles was determined by opticalmicroscopy using a software-based determination of size. Particle sizeof ground materials and micro particles was measured at 1000 foldmagnification by estimation of relative diameter. Distribution wascalculated by comparison of diameter sizes of 1000-5000 particles at 1%v/v. Results are shown in Table 3.

Ligand d (mm) q BSA Type anion exchanger AMBERLITE ® IRA-400 —N⁺—(CH₃)₃(Type 1) 0.3-1.2 0.3 ± 0.11 AMBERLITE ® IRA-743 Methylglucamine 0.5-0.76.1 ± 0.25 DOWEX ® 1X2-100 —N⁺—(CH₃)₃ (Type 1) 0.1-0.5 0.5 ± 0.18DOWEX ® 1X2-400 —N⁺—(CH₃)₃ (Type 1) 0.04-0.07 0.8 ± 0.01 DOWEX ® 1X8-100—N⁺—(CH₃)₃ (Type 1) 0.1-0.5 0.4 ± 0.03 MARATHON ® A2—N⁺—(CH₂—CH₂OH)—(CH₃)₂ (Type 2) 0.4-0.6 0.2 ± 0.02 DIAION ® SA20ADimethylethanolamine 0.3-1.18 n.a. DIAION ® SA10A Trimethylamine0.3-1.18 n.a. DIAION ® SA312 Trimethylamine 0.3-1.18 n.a. Type cationexchanger DOWEX ® 50 WX2-100 —SO₃ ⁻ n.a. n.a. DOWEX ® 50 WX8-100 —SO₃ ⁻n.a. n.a. MARATHON ® C —SO₃ ⁻ 1.2 n.a. MARATHON ® MSC —SO₃ ⁻ 1.2 n.a.DIAION ® PK216 Sulphonic 0.3-1.18 n.a. DIAION ® SK110 Sulphonic 0.3-1.18n.a.

Table 3 lists resins used for preparation of microparticles.

What is claimed is:
 1. A method of manufacturing a protein of interestcomprising: (a) cultivating a host cell that produces the protein ofinterest in a biological fluid; and (b) recovering the protein ofinterest from the biological fluid by: (i) adding to the biologicalfluid positively-charged microparticles comprising a ground polymericanion exchange resin or negatively-charged microparticles comprising aground polymeric cation exchange resin, and (ii) recovering the proteinsof interest from the biological fluid; wherein a mixing of themicroparticles with the biological fluid causes a disruption of thecells or extraction of the proteins of interest from the cells.
 2. Themethod of claim 1, wherein the method further comprises: (c) purifyingthe protein of interest.
 3. The method of claim 2, wherein the methodfurther comprises modifying the protein of interest, preparing apharmaceutical formulation comprising the protein of interest, or both.4. The method of claim 1, wherein the protein of interest is arecombinant protein.
 5. The method of claim 1, wherein the host cellexpresses the protein of interest.
 6. The method of claim 1, wherein thebiological fluid is a cell culture, cell suspension, fermentation broth,culture broth, fermentation supernatant, culture supernatant, or cellsupernatant.
 7. The method of claim 1, wherein the microparticles formflocculus.
 8. The method of claim 1, wherein the anion-exchange resinand the cation exchange resin are polystyrene-based, Hydroxyethylmethacrylate (HEMA)-based, dimethylamino ethylmethacrylate(DMAEMA)-based, dimethylamino ethylmethacrylate (pDMAEMA),polyacrylamide based, or methacrylic acid (MAA)-based.
 9. The method ofclaim 1, wherein the cation exchange resin and anion-exchange resin arepolystyrene cross-linked with divinylbenzene.
 10. The method of claim 1,wherein the microparticles have an average particle size of less thanabout 5 μm.
 11. The method of claim 1, wherein the positively-chargedmicroparticles or negatively-charged microparticles are produced bygrinding a polymeric anion-exchange or cation-exchange resin,respectively.
 12. The method of claim 1, wherein the cells areeukaryotic or prokaryotic cells.
 13. The method of claim 1, furthercomprising allowing the microparticles to form a flocculus beforerecovering the proteins of interest from the biological fluids.
 14. Themethod of claim 13, wherein recovering the proteins of interest from thebiological fluids in step (b)(ii) comprises removing the flocculus fromthe biological fluids and desorbing the proteins of interest from theflocculus.